Morpholine dopamine agonists

ABSTRACT

Compounds of formula (I), (Ia) and (Ib) 
                         
useful as a medicament.

This application claims priority from United Kingdom Application No.0228787.8 filed on Dec. 10, 2002; United Kingdom Application No.0308460.5 filed on Apr. 11, 2003; United Kingdom Application No.0313606.6 filed on Jun. 12, 2003; and U.S. Provisional Application No.60/438,476 filed on Jan. 7, 2003; U.S. Provisional Application No.60/470,950 filed on May 15, 2003 and U.S. Provisional Application No.60/501,512 filed on Sep. 8, 2003.

The present invention relates to a class of dopamine agonists, moreparticularly a class of agonists that are selective for D3 over D2.These compounds are useful for the treatment and/or prevention of sexualdysfunction, for example female sexual dysfunction (FSD), in particularfemale sexual arousal disorder (FSAD) and male sexual dysfunction, inparticular male erectile dysfunction (MED). Male sexual dysfunction asreferred to herein is meant to include ejaculatory disorders such aspremature ejaculation, anorgasmia (inability to achieve orgasm) ordesire disorders such as hypoactive sexual desire disorder (HSDD; lackof interest in sex). These compounds are also useful in treatingneuropsychiatric disorders and neurodegenerative disorders.

The present invention provides for compounds of formula (I), (Ia) and(Ib)

Wherein:

-   A is selected from C—X and N,-   B is selected from C—Y and N,-   R¹ is selected from H and (C₁-C₆)alkyl,-   R² is selected from H and (C₁-C₆)alkyl,-   X is selected from H, HO, C(O)NH₂, NH₂-   Y is selected from H, HO, NH₂, Br, Cl and F-   Z is selected from H, HO, F, CONH₂ and CN;-   And pharmaceutically acceptable salts, solvates and prodrugs    thereof;

With the provisos that:

-   for a compound of formula (I), (Ia) or (Ib), when A is C—X, B is    C—Y, R¹ is H or (C₁-C₆)alkyl and R² is H or (C₁-C₆)alkyl at least    one of X, Y and Z must be OH;-   for a compound of formula (I), when A is C—X and B is C—Y, Y is H, Z    is H, R¹ is H and R² is H, then X cannot be OH.

The pharmaceutically acceptable salts of the compounds of the formula(I) include the acid addition and the base salts thereof.

A pharmaceutically acceptable salt of a compound of the formula (I) maybe readily prepared by mixing together solutions of a compound of theformula (I) and the desired acid or base, as appropriate. The salt mayprecipitate from solution and be collected by filtration or may berecovered by evaporation of the solvent.

Suitable acid addition salts are formed from acids which form non-toxicsalts and examples are the hydrochloride, hydrobromide, hydroiodide,sulphate, bisulphate, nitrate, phosphate, hydrogen phosphate, acetate,maleate, fumarate, lactate, tartrate, citrate, gluconate, succinate,saccharate, benzoate, methanesulphonate, ethanesulphonate,benzenesulphonate, p-toluenesulphonate and pamoate salts.

Suitable base salts are formed from bases which form non-toxic salts andexamples are the sodium, potassium, aluminium, calcium, magnesium, zincand diethanolamine salts.

For a review on suitable salts see Berge et al, J. Pharm. Sci., 66,1-19, 1977.

The pharmaceutically acceptable solvates of the compounds of the formula(I) include the hydrates thereof.

Also included within the present scope of the compounds of the formula(I) are polymorphs thereof.

A compound of the formula (I) contains one or more asymmetric carbonatoms and therefore exists in two or more stereoisomeric forms.

Separation of diastereoisomers may be achieved by conventionaltechniques, e.g. by fractional crystallisation, chromatography orH.P.L.C. of a stereoisomeric mixture of a compound of the formula (I) ora suitable salt or derivative thereof. An individual enantiomer of acompound of the formula (I) may also be prepared from a correspondingoptically pure intermediate or by resolution, such as by H.P.L.C. of thecorresponding racemate using a suitable chiral support or by fractionalcrystallisation of the diastereoisomeric salts formed by reaction of thecorresponding racemate with a suitable optically active acid or base, asappropriate.

Preferred compounds of the present invention are compounds of formula(Ia) and (Ib)

Particularly preferred are compounds of formula (Ia)

Preferably A is C—X or N and B is C—Y

More preferably A is N and B is C—Y

More preferably A is C—X and B is C—Y

Preferably R¹ is selected from H and (C₁-C₄)alkyl.

More preferably R¹ is H, methyl and ethyl

Even more preferably R¹ is H or methyl.

Most preferably R¹ is H.

Preferably R² is selected from H and (C₁-C₄)alkyl.

More preferably R² is selected from H, methyl and ethyl.

Most preferably R² is selected from H and methyl.

In a particularly preferred embodiment R² is H

In a further particularly preferred embodiment R² is methyl

Preferably X is selected from H, OH and NH₂

Most preferably X is selected from H and OH.

In a particularly preferred embodiment X is H

In a further particularly preferred embodiment X is OH

Preferably Y is selected from H, NH₂, Cl and F

Most preferably Y is selected from H and NH₂.

In a particularly preferred embodiment Y is H

In a further particularly preferred embodiment Y is NH₂

Preferably Z is selected from H, HO and F.

Most preferably Z is selected from H or HO.

In a particularly preferred embodiment Z is H

In a further particularly preferred embodiment Z is HO

Particularly preferred are compounds (and salts thereof) of the presentinvention exemplified herein; more preferred are:

R-(−)-3-(4-Propylmorpholin-2-yl)phenol (Example 7A)S-(+)-3-(4-Propylmorpholin-2-yl)phenol (Example 7B)R-(−)-3-(4-Propylmorpholin-2-yl)phenol hydrochloride (Example 8)R-5-(4-Propylmorpholin-2-yl)benzene-1,3-diol (Example 15A)S-5-(4-Propylmorpholin-2-yl)benzene-1,3-diol (Example 15B)R-(+)-2-Fluoro-5-(4-propylmorpholin-2-yl)phenol (Example 23A)S-(−)-2-Fluoro-5-(4-propylmorpholin-2-yl)phenol (Example 23B)2-Bromo-4-(4-propylmorpholin-2-yl)phenol (Example 30)2-Hydroxy-5-(4-propylmorpholin-2-yl)benzamide (Example 35)2-Nitro-4-(4-propylmorpholin-2-yl)phenol (Example 36)2-Amino-4-(4-propylmorpholin-2-yl)phenol (Example 37)5-(4-Propylmorpholin-2-yl)pyridin-2-ylamine (Example 44A and 44B)2-Chloro-5-(4-propyl-morpholin-2-yl)phenol (Example 54)3-[(5S)-5-methyl-4-propylmorpholin-2-yl]phenol (Example 60)5-[(2S,5S)-5-methyl-4-propylmorpholin-2- (Example 66) yl]pyridin-2-amine5-[(2R,5S)-5-methyl-4-propylmorpholin-2- (Example 67) yl]pyridin-2-amine

Most preferred are:

R-(−)-3-(4-Propylmorpholin-2-yl)phenol (Example 7A)S-(+)-3-(4-Propylmorpholin-2-yl)phenol (Example 7B)R-(−)-3-(4-Propylmorpholin-2-yl)phenol hydrochloride (Example 8)R-5-(4-Propylmorpholin-2-yl)benzene-1,3-diol (Example 15A)S-5-(4-Propylmorpholin-2-yl)benzene-1,3-diol (Example 15B)R-(+)-2-Fluoro-5-(4-propylmorpholin-2-yl)phenol (Example 23A)S-(−)-2-Fluoro-5-(4-propylmorpholin-2-yl) phenol (Example 23B)5-(4-Propylmorpholin-2-yl)pyridin-2-ylamine (Example 44A and 44B)2-Chloro-5-(4-propyl-morpholin-2-yl)phenol (Example 54)5-[(2S,5S)-5-methyl-4-propylmorpholin-2- (Example 66) yl]pyridin-2-amine5-[(2R,5S)-5-methyl-4-propylmorpholin-2- (Example 67) yl]pyridin-2-amine

Compounds of the invention may be prepared, in known manner, in avariety of ways. The routes below illustrate methods of synthesisingcompounds of formula (I); the skilled man will appreciate that compoundsof formula (Ia) and (Ib) may be isolated with appropriate resolutiontechniques.

Compounds of general formula I where A is C—X, B is C—Y, R¹ is H or(C₁-C₆)alkyl, R² is H and where X, Y and Z are as described herein maybe prepared according to reaction scheme 1.

Compounds of formula (III) may be prepared by reacting an aldehyde offormula II with i) a cyanide source or nitromethane followed by ii)reduction with borane, lithium aluminium hydride or hydrogenation. Somecompounds of formula II and III are also commercially available.

Compounds of formula (IV) may be prepared by reacting compounds offormula (III) with iii), acid chlorides in the presence of a suitablebase such as triethylamine or 4-methylmorpholine. Typical reactionconditions comprise 1.0 equivalents of amine (III), 1.2-2.0 equivalentsof base (preferably triethylamine), 1.1-1.3 equivalents of acid chloridein dichloromethane at 25° C.

Compounds of formula (V) may be prepared by reducing compounds offormula (IV) with iv), reducing agents such as borane or lithiumaluminium hydride. Typical conditions comprise 1.0 equivalents of amide(IV), 1.2-3.0 equivalents of borane in THF at reflux. Compounds offormula (V) can also be made by reductive animation of compounds offormula (III) with a suitable aldehyde in the presence of sodiumcyanoborohydride.

Compounds of formula (VI) may be prepared by reacting compounds offormula V with v), chloroacetyl chloride or 2-substituted chloroacetylchlorides (such as 2-chloropropionyl chloride or 2-chlorobutyrylchloride) in the presence of base such as triethylamine, sodiumcarbonate and potassium hydroxide. Typical conditions comprise 1.0equivalents of amine IV, 1.0-1.3 equivalents of acid chloride, 1.2-2.0equivalents of triethylamine in dichloromethane at 25° C., the crudereaction mixture is then dissolved in IPA with 1.2-3.0 equivalents ofaqueous potassium hydroxide.

Compounds of formula (I) may be prepared by reacting compounds offormula (VI) with vi), reducing agents such as borane or lithiumaluminium hydride. Typical conditions comprise 1.0 equivalents of amideVI, 1.2-3.0 equivalents of borane in THF at reflux.

The skilled man will appreciate that due to one of X, Y or Z being ahydroxy group, it will be necessary to protect the hydroxy group(s) witha suitable protecting group throughout the transformations of scheme 1,then remove the protecting group. Methods for deprotection of a phenolgroup depend on the protecting group. For examples ofprotection/deprotection methodology see “Protective groups in Organicsynthesis”, T W Greene and P G M Wutz. For example, where the hydroxy isprotected as a methyl ether, deprotection conditions comprise refluxingin 48% aqueous HBr for 1-24 hours, or by stirring with borane tribromidein dichloromethane for 1-24 hours. Alternatively where the hydroxy isprotected as a benzyl ether, deprotection conditions comprisehydrogenation with a palladium catalyst under a hydrogen atmosphere.

Compounds of general formula (I) where one of A or B is N, R¹ is H or(C₁-C₆)alkyl, R² is H and X, Y, and Z are as described herein, with theproviso that one of X, Y or Z is NH₂, may be prepared according toreaction scheme 2. Scheme is illustrated where B is C—Y and where Y isNH₂; the skilled man will understand that the alternative compounds areequally practicable.

Compounds of formula (VII) may be prepared using the process asdescribed in JP2001048864.

Compounds of formula (VIII) may be prepared by reacting epoxide (VII)with vii), propylamine. Typical reaction conditions comprise stirringthe epoxide with excess amine either neat or in dimethylsulphoxide.

Compounds of formula (IX) may be prepared by reacting compounds offormula (VIII) with v), chloroacetyl chloride or 2-substitutedchloroacetyl chlorides (such as 2-chloropropionyl chloride or2-chlorobutyryl chloride) in the presence of base such as triethylamine,sodium carbonate and potassium hydroxide. Typical conditions comprise1.0 equivalents of amine (VIII), 1.2-2.0 equivalents of triethylamine indichloromethane at 25° C., the crude reaction mixture is then dissolvedin IPA with 1.2-3.0 equivalents of aqueous potassium hydroxide.

Compounds of formula (X) may be prepared by reacting compounds offormula (IX) with reducing agents such as lithium aluminium hydride.Typical conditions comprise 1.0 equivalents of amide (X), 1.2equivalents of lithium aluminium hydride in THF at reflux.

Compounds of formula (I) may be prepared by ix), deprotection. Typicalconditions comprise 1.0 equivalents of compound X and 5 equivalents ofhydroxylamine hydrochloride in ethanol at reflux.

Compounds of general formula I, where A is C—X, B is C—Y, R¹ is H and R²is H or (C₁-C₆) alkyl and where X, Y and Z are as described herein maybe prepared according to reaction scheme 3.

Compounds of the formula (XII) may be prepared by reacting an amino acidester of the formula (XI) with x) acid chlorides in the presence of asuitable base such as triethylamine and 4-methylmorpholine. Typicalreaction conditions comprise 1 equivalent amino acid ester (XI), 1equivalent of acid chloride and 3 equivalents of base in dichloromethaneat 25° C. Some compounds of formula (XI) are commercially available.

Compounds of the formula (XIII) may be prepared by reacting compounds ofthe formula (XII) with xi) borane-THF complex, with subsequent breakingof the boron-nitrogen complex with acid and t-butyloxycarbonylprotection of the formed amine. Typical reaction conditions comprise 1equivalent of the amide (XII) with 3 equivalents of BH₃-THF in THF atreflux, cooling, cautious addition of 6M aqueous HCl, and heating toreflux for a further 6 h.

Subsequent evaporation of solvent, redissolution in a methanol:water(8:1) mix, and addition of 5 equivalents of a base such as potassiumhydroxide and 1.5 equivalents of di-tert-butyl dicarbonate, and stirringof the mixture for 72 hours.

Compounds of the formula (XIV) may be prepared by reacting compounds ofthe formula (XIII) with xii) an organic solution of HCl. Typicalreaction conditions comprise 1 equivalent of the carbamate (XIII) and a1-10 equivalents of a 4M solution of HCl in dioxan in dioxan at 25° C.

Compounds of the formula (XV) may be prepared by reacting compounds ofthe formula (XIV) with xiii) a 2-bromoacetophenone in the presence of abase such as triethylamine or 4-methylmorpholine. The2-bromoacetophenones may be obtained from commerical sources oralternatively prepared from the parent acetophenone by standardbromination methodology well known to those skilled in the art. Typicalconditions comprise 1 equivalent of the aminoalcohol (XIV) with 1-3equivalents of triethylamine and 1 equivalent of a 2-bromoacetophenoneat 65° C.

Compounds of the formula (I) may be prepared by reacting compounds ofthe formula (XV) with xiv) triethylsilane and trimethylsilyltriflate.Typical conditions comprise addition of 5-10 equivalents oftriethylsilane to 1 equivalent of the morpholinol (XV) indichloromethane at −78° C. followed by addition of 2 equivalents oftrimethylsilyltriflate.

The skilled man will appreciate that due to one of X, Y or Z being ahydroxy group, it will be necessary to protect the hydroxy group(s) witha suitable protecting group throughout the transformations of scheme 3,then remove the protecting group. Methods for deprotection of a phenolgroup depend on the protecting group. For examples ofprotection/deprotection methodology see “Protective groups in Organicsynthesis”, T W Greene and P G M Wutz. For example, where the hydroxy isprotected as a methyl ether, deprotection conditions comprise refluxingin 48% aqueous HBr for 1-24 hours, or by stirring with borane tribromidein dichloromethane for 1-24 hours. Alternatively where the hydroxy isprotected as a benzyl ether, deprotection conditions comprisehydrogenation with a palladium catalyst under a hydrogen atmosphere.

Compounds of the formula (I) where the stereocentre alpha to themorpholine nitrogen is defined absolutely may be prepared starting fromhomochiral compounds of the formula (XI), which may be commerciallyavailable or obtained through methods readily available to the skilledman in the chemistry literature. The resulting compounds of the formula(I) will contain a mixture of diastereoisomers which may be separated onan HPLC column. Typical conditions comprise eluting through a ChiralcelOJ-H column with 100% MeOH mobile phase.

Compounds of general formula (I) where one of A or B is N, R¹ is H, R²is H or (C₁-C₆)alkyl and X, Y and Z are as described herein, with theproviso that one of X, Y or Z is NH₂, may be prepared according toreaction scheme 4. The scheme is illustrated where B is C—Y and where Yis NH₂; the skilled artisan will understand that the alternativecompounds are equally practicable.

Compounds of formula (XVIII) may be prepared by reacting compounds offormula (XVI) with xv) amino alcohols of formula (XIV) in the presenceof a base such as triethylamine or 4-methylmorpholine. Typicalconditions comprise 1 equivalent of the aminoalcohol (XIV) with 1-3equivalents of triethylamine and 1 equivalent of a compound of formula(XVI) using toluene as solvent at room temperature or above. Compoundsof formula (XVI) are commercially available.

Compounds of formula (IXX) may be prepared by reacting a compound offormula (XVIII) with xvi) an organometallic reagent formed from thebromide of formula (XVII). Suitable organometallic reagents includeGrignard (organomagnesium) or organolithium reagents, which may beprepared from the bromide by halogen metal exchange. Typical conditionscomprise addition of isopropylmagensium chloride to the bromide (XVII)in an anhydrous ethereal solvent such as tetrahydrofuran at roomtemperature (to perform the halogen metal exchange reaction), followedby addition of the morpholinone (XVIII). The bromide (XVII) may beprepared using the process as described in WO9932475.

Morpholinol (IXX) may be reduced to diol (XX) by xvii) reaction with ahydride reducing agent, such as sodium borohydride in an alcohol solventsuch as methanol.

Compounds of formula (XXI) may be prepared from the diol (XX) by ix),deprotection. Typical conditions comprise 1.0 equivalents of compound(XX) and 5 equivalents of hydroxylamine hydrochloride in ethanol atreflux.

Compounds of formula (I) may be prepared by xviii) cyclisation ofcompounds of formula (XXI) by treatment with acid. Typical conditionsemploy concentrated sulfuric acid and dichloromethane as solvent at roomtemperature or above.

All of the above reactions and the preparations of novel startingmaterials using in the preceding methods are conventional andappropriate reagents and reaction conditions for their performance orpreparation as well as procedures for isolating the desired productswill be well-known to those skilled in the art with reference toliterature precedents and the Examples and Preparations hereto.

The compounds of the present invention have utility as selective D3agonists in the treatment of disease states. There are a number ofcompounds with activity as both D2 and D3 agonists; however the use ofsuch compounds is associated with a large number of side effectsincluding nausea, emesis, syncope, hypotension and bradycardia, some ofwhich are a cause for serious concern.

It was previously held that the efficacy of the prior art compoundsstemmed from their ability to agonise D2; however D2 agonism isimplicated as a cause of the side effects detailed above.

The present invention provides a class of selective D3 agonists.Serendipitously, these have been found to be efficacious, whilstreducing the side effects associated with unselective prior artcompounds.

Compounds of present invention are useful in treating sexualdysfunction, female sexual dysfunction, including hypoactive sexualdesire disorder, sexual arousal disorder, orgasmic disorder and sexualpain disorder; male erectile dysfunction, hypertension,neurodegeneration, psychiatric disorders, depression (e.g. depression incancer patients, depression in Parkinson's patients, postmyocardialinfarction depression, subsyndromal symptomatic depression, depressionin infertile women, paediatric depression, major depression, singleepisode depression, recurrent depression, child abuse induceddepression, post partum depression and grumpy old man syndrome),generalized anxiety disorder, phobias (e.g. agoraphobia, social phobiaand simple phobias), posttraumatic stress syndrome, avoidant personalitydisorder, premature ejaculation, eating disorders (e.g. anorexia nervosaand bulimia nervosa), obesity, chemical dependencies (e.g. addictions toalcohol, cocaine, heroin, phenobarbital, nicotine and benzodiazepines),cluster headache, migraine, pain, Alzheimer's disease,obsessive-compulsive disorder, panic disorder, memory disorders (e.g.dementia, amnestic disorders, and age-related cognitive decline (ARCD)),Parkinson's diseases (e.g. dementia in Parkinson's disease,neuroleptic-induced parkinsonism and tardive dyskinesias), endocrinedisorders (e.g. hyperprolactinaemia), vasospasm (particularly in thecerebral vasculature), cerebellar ataxia, gastrointestinal tractdisorders (involving changes in motility and secretion), negativesymptoms of schizophrenia, premenstrual syndrome, fibromyalgia syndrome,stress incontinence, Tourette's syndrome, trichotillomania, kleptomania,male impotence, attention deficit hyperactivity disorder (ADHD), chronicparoxysmal hemicrania, headache (associated with vascular disorders),emotional lability, pathological crying, sleeping disorder (cataplexy)and shock.

Compounds of the present invention are particularly suitable fortreating female sexual dysfunction, male erectile dysfunction,neurodegeneration, depression and psychiatric disorders.

The compounds of the present invention are useful in male sexualdysfunction, particularly male erectile dysfunction. Male erectiledysfunction (MED), otherwise known as male erectile disorder, is definedas:

-   -   “the inability to achieve and/or maintain a penile erection for        satisfactory sexual performance” (NIH Consensus Development        Panel on Impotence, 1993)”

It has been estimated that the prevalence of erectile dysfunction (ED)of all degrees (minimal, moderate and complete impotence) is 52% in men40 to 70 years old, with higher rates in those older than 70 (Melman etal 1999, J. Urology, 161, p 5-11). The condition has a significantnegative impact on the quality of life of the individual and theirpartner, often resulting in increased anxiety and tension which leads todepression and low self-esteem. Whereas two decades ago, MED wasprimarily considered to be a psychological disorder (Benet et al 1994Comp. Ther., 20: 669-673), it is now known that for the majority ofindividuals there is an underlying organic cause. As a result, muchprogress has been made in identifying the mechanism of normal penileerection and the pathophysiologies of MED.

Penile erection is a haemodynamic event which is dependent upon thebalance of contraction and relaxation of the corpus cavernosal smoothmuscle and vasculature of the penis (Lerner et al 1993, J. Urology, 149,1256-1255). Corpus cavernosal smooth muscle is also referred to hereinas corporal smooth muscle or in the plural sense corpus cavernosa.Relaxation of the corpus cavernosal smooth muscle leads to an increasedblood flow into the trabecular spaces of the corpus cavernosa, causingthem to expand against the surrounding tunica and compress the drainingveins. This produces a vast elevation in blood pressure which results inan erection (Naylor, 1998, J. Urology, 81, 424-431).

The changes that occur during the erectile process are complex andrequire a high degree of co-ordinated control involving the peripheraland central nervous systems, and the endocrine system (Naylor, 1998, J.Urology, 81, 424-431). Corporal smooth muscle contraction is modulatedby sympathetic noradrenergic innervation via activation of postsynapticα₁ adrenoceptors. MED may be associated with an increase in theendogenous smooth muscle tone of the corpus cavernosum. However, theprocess of corporal smooth muscle relaxation is mediated partly bynon-adrenergic, non-cholinergic (NANC) neurotransmission. There are anumber of other NANC neurotransmitters found in the penis, other thanNO, such as calcitonin gene related peptide (CGRP) and vasoactiveintestinal peptide (VIP). The main relaxing factor responsible formediating this relaxation is nitric oxide (NO), which is synthesisedfrom L-arginine by nitric oxide synthase (NOS) (Taub et al 1993 Urology,42, 698-704). It is thought that reducing corporal smooth muscle tonemay aid NO to induce relaxation of the corpus cavernosum. During sexualarousal in the male, NO is released from neurones and the endotheliumand binds to and activates soluble guanylate cyclase (sGC) located inthe smooth muscle cells and endothelium, leading to an elevation inintracellular cyclic guanosine 3′,5′-monophosphate (cGMP) levels. Thisrise in cGMP leads to a relaxation of the corpus cavernosum due to areduction in the intracellular calcium concentration ([Ca²⁺]_(i)), viaunknown mechanisms thought to involve protein kinase G activation(possibly due to activation of Ca²⁺ pumps and Ca²⁺-activated K⁺channels).

Multiple potential sites have been identified within the central nervoussystem for the modulation of sexual behaviour. The key neurotransmittersare thought to be serotonin, norepinephrine, oxytocin, nitric oxide anddopamine. By mimicking the actions of one of these key neurotransmitterssexual function may be adjusted. Dopamine D3 receptors are expressedalmost exclusively in the limbic are of the brain, regions involved inthe reward, emotional and cognitive processes.

Without being bound by any theory, it appears that “due to its role inthe control of locomotor activity, the integrity of the nigrostriataldopaminergic pathway is also essential for the display of copulatorybehaviour. Somehow, more specific to sexual function, it is likely thatdopamine can trigger penile erection by acting on oxytocinergic neuronslocated in the paraventricular nucleus of the hypothalamus, and perhapson the pro-erectile sacral parasympathetic nucleus within the spinalcord”. It now appears that the significant site is D3 and not aspreviously thought, D2.

In essence, D3 is an initiator of sexual behaviour.

Accordingly, the present invention provides for, the use of a compoundof formula (I) in the preparation of a medicament for the treatment orprevention of erectile dysfunction.

Patients with mild to moderate MED should benefit from treatment withthe compounds according to the present invention, and patients withsevere MED may also respond. However, early investigations suggest thatthe responder rate of patients with mild, moderate and severe MED may begreater with a selective D3 agonist/PDE5 inhibitor combination. Mild,moderate and severe MED will be terms known to the man skilled in theart, but guidance can be found in The Journal of Urology, vol. 151,54-61 (Jan 1994).

Early investigations suggest the below mentioned MED patient groupsshould benefit from treatment with a selective D3 agonist and a PDE5i(or other combination set out hereinafter). These patient groups, whichare described in more detail in Clinical Andrology vol. 23, no. 4, p773-782 and chapter 3 of the book by I. Eardley and K. Sethia “ErectileDysfunction-Current Investigation and Management, published byMosby-Wolfe, are as follows: psychogenic, organic, vascular,endocrinologic, neurogenic, arteriogenic, drug-induced sexualdysfunction (lactogenic) and sexual dysfunction related to cavernosalfactors, particularly venogenic causes. Accordingly the presentinvention provides for the use of a compound of formula (I), (Ia) or(Ib) in the preparation of a medicament in combination with a PDE5inhibitor for the treatment of erectile dysfunction.

Suitable PDE5 inhibitors are described herein.

The compounds of the present invention are useful in the treatment orprevention of female sexual dysfunction (FSD), particularly FSAD.

In accordance with the invention, FSD can be defined as the difficultyor inability of a woman to find satisfaction in sexual expression. FSDis a collective term for several diverse female sexual disorders(Leiblum, S. R. (1998)—Definition and classification of female sexualdisorders. Int. J. Impotence Res., 10, S104-S106; Berman, J. R., Berman,L. & Goldstein, I. (1999)—Female sexual dysfunction: Incidence,pathophysiology, evaluations and treatment options. Urology, 54,385-391.). The woman may have lack of desire, difficulty with arousal ororgasm, pain with intercourse or a combination of these problems.Several types of disease, medications, injuries or psychologicalproblems can cause FSD. Treatments in development are targeted to treatspecific subtypes of FSD, predominantly desire and arousal disorders.

The categories of FSD are best defined by contrasting them to the phasesof normal female sexual response: desire, arousal and orgasm (Leiblum,S. R. (1998)—Definition and classification of female sexual disorders.Int. J. Impotence Res., 10, S104-S106). Desire or libido is the drivefor sexual expression. Its manifestations often include sexual thoughtseither when in the company of an interested partner or when exposed toother erotic stimuli. Arousal is the vascular response to sexualstimulation, an important component of which is genital engorgement andincludes increased vaginal lubrication, elongation of the vagina andincreased genital sensation/sensitivity. Orgasm is the release of sexualtension that has culminated during arousal.

Hence, FSD occurs when a woman has an inadequate or unsatisfactoryresponse in any of these phases, usually desire, arousal or orgasm. FSDcategories include hypoactive sexual desire disorder, sexual arousaldisorder, orgasmic disorders and sexual pain disorders. Although thecompounds of the invention will improve the genital response to sexualstimulation (as in female sexual arousal disorder), in doing so it mayalso improve the associated pain, distress and discomfort associatedwith intercourse and so treat other female sexual disorders.

Hypoactive sexual desire disorder is present if a woman has no or littledesire to be sexual, and has no or few sexual thoughts or fantasies.This type of FSD can be caused by low testosterone levels, due either tonatural menopause or to surgical menopause. Other causes includeillness, medications, fatigue, depression and anxiety.

Female sexual arousal disorder (FSAD) is characterised by inadequategenital response to sexual stimulation. The genitalia do not undergo theengorgement that characterises normal sexual arousal. The vaginal wallsare poorly lubricated, so that intercourse is painful. Orgasms may beimpeded. Arousal disorder can be caused by reduced oestrogen atmenopause or after childbirth and during lactation, as well as byillnesses, with vascular components such as diabetes andatherosclerosis. Other causes result from treatment with diuretics,antihistamines, antidepressants e.g. selective serotonin re-uptakeinhibitors (SSRIs) or antihypertensive agents.

Sexual pain disorders (includes dyspareunia and vaginismus) ischaracterised by pain resulting from penetration and may be caused bymedications which reduce lubrication, endometriosis, pelvic inflammatorydisease, inflammatory bowel disease or urinary tract problems.

As previously discussed, D3 is thought to be an initiator of sexualbehaviour. The clitoris is considered to be a homologue of the penis(Levin, R. J. (1991), Exp. Clin. Endocrinol., 98, 61-69); the samemechanism that provides provides an erectile response in the maleproduces an increase in genital blood flow in the female with anassociated effect upon FSD. In addition there are changes inproceptivity and receptivity.

Thus, in accordance with a preferred aspect of the invention, there isprovided use of a compound of formula (I), (Ia) or (Ib) in thepreparation of a medicament for the treatment or prophylaxis of femalesexual dysfunction, more particularly hypoactive sexual desire disorder,sexual arousal disorder, orgasmic disorder and sexual pain disorder.

Preferably the compounds of formula (I) are useful in the treatment orprophylaxis of sexual arousal disorder, orgasmic disorder, andhypoactive sexual desire disorder, and most preferably in the treatmentor prophylaxis of sexual arousal disorder.

In a preferred embodiment the compounds of formula (I), (Ia) and (Ib)are useful in the treatment of a subject with female sexual arousaldisorder and concomitant hypoactive sexual desire disorder.

The Diagnostic and Statistical Manual (DSM) IV of the AmericanPsychiatric Association defines Female Sexual Arousal Disorder (FSAD) asbeing:

“. . . a persistent or recurrent inability to attain or to maintainuntil completion of the sexual activity adequate lubrication-swellingresponse of sexual excitement. The disturbance must cause markeddistress or interpersonal difficulty . . . ”.

The arousal response consists of vasocongestion in the pelvis, vaginallubrication and expansion and swelling of the external genitalia. Thedisturbance causes marked distress and/or interpersonal difficulty.

FSAD is a highly prevalent sexual disorder affecting pre-, peri- andpost-menopausal (±hormone replacement therapy (HRT)) women. It isassociated with concomitant disorders such as depression, cardiovasculardiseases, diabetes and urogenital (UG) disorders.

The primary consequences of FSAD are lack of engorgement/swelling, lackof lubrication and lack of pleasurable genital sensation. The secondaryconsequences of FSAD are reduced sexual desire, pain during intercourseand difficulty in achieving an orgasm.

It has recently been hypothesised that there is a vascular basis for atleast a proportion of patients with symptoms of FSAD (Goldstein et al.,Int. J. Impot. Res., 10, S84-S90, 1998) with animal data supporting thisview (Park et al., Int. J. Impot. Res., 9, 27-37, 1997).

R. J. Levin teaches us that because “. . . male and female genitaliadevelop embryologically from the common tissue anlagen, [that] male andfemale genital structures are argued to be homologues of one another.Thus the clitoris is the penile homologue and the labia homologues ofthe scrotal sac. . . . ” (Levin, R. J. (1991), Exp. Clin. Endocrinol.,98, 61-69).

Drug candidates for treating FSAD, which are under investigation forefficacy, are primarily erectile dysfunction therapies that promotecirculation to male genitalia.

The compounds of the present invention are advantageous by providing ameans for restoring a normal sexual arousal response—namely increasedgenital blood flow leading to vaginal, clitoral and labial engorgement.This will result in increased vaginal lubrication via plasmatransudation, increased vaginal compliance and increased genitalsensitivity. Hence, the present invention provides a means to restore,or potentiate, the normal sexual arousal response.

Thus, in accordance with a preferred aspect of the invention, there isprovided use of a compound of formula (I), (Ia) or (Ib) in thepreparation of a medicament for the treatment or prophylaxis of femalesexual arousal disorder.

By female genitalia herein we mean: “The genital organs consist of aninternal and external group. The internal organs are situated within thepelvis and consist of ovaries, the uterine tubes, uterus and the vagina.The external organs are superficial to the urogenital diaphragm andbelow the pelvic arch. They comprise the mons pubis, the labia majoraand minora pudendi, the clitoris, the vestibule, the bulb of thevestibule, and the greater vestibular glands” (Gray's Anatomy, C. D.Clemente, 13^(th) American Edition).

The compounds of the invention find application in the followingsub-populations of patients with FSD: the young, the elderly,pre-menopausal, peri-menopausal, post-menopausal women with or withouthormone replacement therapy.

The compounds of the invention find application in patients with FSDarising from:-

-   i) Vasculogenic etiologies e.g. cardiovascular or atherosclerotic    diseases, hypercholesterolemia, cigarette smoking, diabetes,    hypertension, radiation and perineal trauma, traumatic injury to the    iliohypogastric pudendal vascular system.-   ii) Neurogenic etiologies such as spinal cord injuries or diseases    of the central nervous system including multiple sclerosis,    diabetes, Parkinsonism, cerebrovascular accidents, peripheral    neuropathies, trauma or radical pelvic surgery.-   iii) Hormonal/endocrine etiologies such as dysfunction of the    hypothalamic/pituitary/gonadal axis, or dysfunction of the ovaries,    dysfunction of the pancreas, surgical or medical castration,    androgen deficiency, high circulating levels of prolactin e.g.    hyperprolactinemia, natural menopause, premature ovarian failure,    hyper and hypothyroidism.-   iv) Psychogenic etiologies such as depression, obsessive compulsive    disorder, anxiety disorder, postnatal depression/“Baby Blues”,    emotional and relational issues, performance anxiety, marital    discord, dysfunctional attitudes, sexual phobias, religious    inhibition or a traumatic past experiences.-   v) Drug-induced sexual dysfunction resulting from therapy with    selective serotonin reuptake inhibitors (SSRis) and other    antidepressant therapies (tricyclics and major tranquillizers),    anti-hypertensive therapies, sympatholytic drugs, chronic oral    contraceptive pill therapy.

The Compounds of the present invention are also useful in the treatmentof depression.

Dopamine D3 receptors are expressed almost exclusively in the limbicarea of the brain, regions involved in reward, emotional and cognitiveprocesses. Chronic treatment with several classes of antidepressants areknown to increase the expression of D3 in the limbic area, andantidepressant effects of desipramine can be blocked by sulpride (D2/D3antagonist) when injected to nucleus accumbens (area rich in D3) but notcaudate-putamen (area rich in dopamine D2 receptors). In addition,antidepressant effects were observed preclinical models of depressionand in patients treated with pramipexole, a D3-preferring agonist. Theavailable information suggests that D3 receptors mediate theanti-depressant activity and that selective D3 receptor agonistsrepresent a new class of antidepressant drugs. Since antidepressants areknown to be effective in other psychiatric disorders, D3 agonists wouldhave the potential to treat psychiatric diseases.

The present invention provides for the use of a selective D3 agonist inthe preparation of a medicament for the treatment of depression andpsychiatric diseases.

Preferably said D3 agonist exhibit a functional potency at D3 receptorexpressed as an EC50, lower than 1000 nM, more preferably lower than 100nM, yet more preferably lower than 50 nM, most preferably lower than 10nM.

Preferably said D3 agonist has a selectivity for D3 over D2, whereinsaid dopamine D3 receptor agonist is at least about 15-times, preferablyat least about 27-times, more preferably at least about 30-times, mostpreferably at least about 100-times more functionally selective for adopamine D3 receptor as compared with a dopamine D2 receptor

Suitable conditions include depression (e.g. depression in cancerpatients, depression in Parkinson's patients, postmyocardial infarctiondepression, subsyndromal symptomatic depression, depression in infertilewomen, paediatric depression, major depression, single episodedepression, recurrent depression, child abuse induced depression, postpartum depression and grumpy old man syndrome), generalized anxietydisorder, phobias (e.g. agoraphobia, social phobia and simple phobias),posttraumatic stress syndrome, avoidant personality disorder, eatingdisorders (e.g. anorexia nervosa and bulimia nervosa), obesity, chemicaldependencies (e.g. addictions to alcohol, cocaine, heroin,phenobarbital, nicotine and benzodiazepines), Alzheimer's disease,obsessive-compulsive disorder, panic disorder, memory disorders (e.g.dementia, amnestic disorders, and age-related cognitive decline (ARCD)),Parkinson's diseases (e.g. dementia in Parkinson's disease,neuroleptic-induced parkinsonism and tardive dyskinesias), endocrinedisorders (e.g. hyperprolactinaemia), vasospasm (particularly in thecerebral vasculature), cerebellar ataxia, negative symptoms ofschizophrenia, stress incontinence, Tourette's syndrome,trichotillomania, kleptomania, attention deficit hyperactivity disorder(ADHD), chronic paroxysmal hemicrania, emotional lability, pathologicalcrying, sleeping disorder (cataplexy) and shock.

In a preferred embodiment, the present invention provides for the use ofa compound of formula (I), (Ia) and (Ib) in the preparation of amedicament for the treatment of depression or psychiatric disorders.

Suitable depressive conditions and psychiatric disorders are describedabove.

The compounds of the present invention also have utility in thetreatment of neurodegeneration; sources of neurodegeneration includeneurotoxin poisoning; vision loss caused by neurodegeneration of thevisual pathway, such as by a stroke in the visual pathway eg in retina,optic nerve and/or occipital lobe; epileptic seizures; and fromimpairment of glucose and/or oxygen supply to the brain.

Accordingly the present invention provides for the use of a selective D3agonist in the preparation of a medicament for the treatment ofneurodegeneration.

Preferably said D3 agonist exhibit a functional potency at D3 receptorexpressed as an EC50, lower than 1000 nM, more preferably lower than 100nM, yet more preferably lower than 50 nM, most preferably lower than 10nM.

Preferably said D3 agonist has a selectivity for D3 over D2, whereinsaid dopamine D3 receptor agonist is at least about 15-times, preferablyat least about 27-times, more preferably at least about 30-times, mostpreferably at least about 100-times more functionally selective for adopamine D3 receptor as compared with a dopamine D2 receptor

In a preferred embodiment, the D3 agonist is a compound of formula (I),(Ia) or (Ib)

In addition to their role in treating Sexual dysfunction, depression,neurodegeneration and psychiatric disorders, the compounds of thepresent invention are likely to be efficacious in a number of additionalindications.

Accordingly, the present invention provides for the use of compounds offormula (I), (Ia) or (Ib) in the preparation of a medicament for thetreatment of hypertension, premature ejaculation, obesity, clusterheadache, migraine, pain, endocrine disorders (e.g.hyperprolactinaemia), vasospasm (particularly in the cerebralvasculature), cerebellar ataxia, gastrointestinal tract disorders(involving changes in motility and secretion), premenstrual syndrome,fibromyalgia syndrome, stress incontinence, trichotillomania and chronicparoxysmal hemicrania, headache (associated with vascular disorders).

D3/D2 Agonist Bind Assay

Gonazalez et al (Eup. J Pharmacology 272 (1995) R1-R3) discloses anassay for determining the binding capability of a compound at D3 and/orD2 dopamine receptors and thus the binding selectivity of suchcompounds. This assay is, thus, herein referred to as a binding assay.

D3/D2 Agonist Functional Assay

A suitable assay for determining functionally the activity of a compoundat D3 and/or D2 dopamine receptors is detailed hereinbelow.

Compounds are evaluated as agonists or antagonists at the dopamine D2and D3 receptors by looking at cAMP levels in a GH4C1 and CHO cell-lineexpressing the human D2 and D3 receptors, respectively.

Experimental Procedures

Inhibition Via Dopamine D3 Receptors of Forskolin-Stimulated AdenylateCyclase Activity

Materials

Cell Culture Media:

hD₃CHO Medium DMEM, high glucose (Sigma D5671) 2mM L-Glutamine (SigmaG7513) 10% dialyzed FBS (Sigma F0392)

hD₃CHO (Chinese hamster Ovary) cells expressing the human Dopamine D3receptor were generated in house. These cells are deficient in thedihydrofolate reductase gene.

-   -   Media is made up fresh every week as below, and filtered through        a 0.22 μM filter before use. Media is stored at 4° C. and warmed        to 37° C. prior to addition to the cells.        Cell Dissociation Solution (CDS): (Sigma C-5914)

5 ml used to harvest cells from 225 cm² flask (37° C. 5 min forhD2LGH4C1 cells and 10 minutes for hD3CHO cells).

Phosphate Buffer d Salin (PBS): (Gibco. 14040-091)

Trypan Blue: (Sigma T8154)

Forskolin (Calbiochem 344273)

Dissolved to a concentration of 20 mM in distilled water. (This stock isstored at +4° C.). 4× assay stock of 40 μM is made by carrying out a500-fold dilution in PBS buffer. 25 μl of the 40 μM stock is added to afinal assay volume of 100 μl, yielding a final assay concentration of 10μM.

Test Compounds

Dissolved in 100% DMSO to yield a stock concentration of 10 mM.

Pramipexole Standard

Dissolved in 100% DMSO to yield a stock concentration of 10 mM.

Cyclase Activation Flashplate Assay (NEN SMP004B)

Supplied by Perkin-Elmer Life Sciences, Inc

[¹²⁵I]-cyclic Adenosine Monophosphate (cAMP) (NEX 130)

-   -   Supplied by Perkin-Elmer Life Sciences, Inc        Specific Equipment

Westbart Microtitre Plate Shaker/Incubator

Packard Topcount NXT (ECADA compatible programme)

Tecan Genesis

Labsystems Multi-drop DW

Protocol Testing Compound Activity with hD₃CHO Cells

Compound Dilutions

-   -   Pramipexole is included as a reference standard. A 10-point,        semi-log curve is generated every 4 plates. Compound results are        normalised to the minimum (0 nM pramipexole) and maximum (100 nM        pramipexole) responses generated by the cells. All test        compounds may also be tested via a 10-point (semi-log) curve.    -   Test compounds are dissolved in 100% DMSO to yield a stock        concentration of 10 mM. These are further diluted in 100% DMSO        to 1 mM via a 10-fold dilution (1000× the final assay        concentration required, e.g. 1 mM will give a top concentration        of 1 μM).    -   Pramipexole is dissolved in 100% DMSO to give a concentration of        10 mM. Pramipexole is diluted further to 0.1 mM in 100% DMSO via        a 100-fold dilution.    -   Further dilutions and additions are carried out in 0.4% DMSO/PBS        using a suitable Tecan Genesis Protocol, capable of performing        serial dilutions at a fold of 3.159 (semi-log unit).

Tecan Genesis Dilutions

-   -   10 μL of the test compounds are added to column 1 of a        microplate. 240 μL of 0.4% DMSO/PBS is added to this to give a        25-fold dilution (0.04 mM). 20 μL of the 0.04 mM dilution is        transferred to the wells of column 2 where 180 μL of 0.4%        DMSO/PBS is added, giving a further 10-fold dilution to achieve        a 4×top assay concentration (0.004 mM).    -   Serial dilutions are performed (3.159-fold) to achieve a        semi-log dilution series:        -   4 μM, 1.27 μM, 400 nM, 127 nM, 40 nM, 13 nM, 4 nM, 1.27 nM,            0.4 nM, 0.1 nM    -   25 μL (in duplicate) of the serial dilutions are transferred to        columns 2-11 of the Flashplate (See Appendix). Since the final        assay volume is 100 μL, the final assay concentrations will be:        -   1000 μM, 317 nM, 100 nM, 32 nM, 10 nM, 3.2 nM, 1 nM, 0.3 nM,            0.1 nM, 0.03 nM    -   Minimum control (low control): 25 μL 0.4% DMSO/PBS (vehicle) is        added to the following wells (column 1 wells E-H and column 2        wells A-D). Cells+forskolin are added later.    -   Maximum control (high control): 10 mM pramipexole is diluted in        PBS via a 250-fold dilution (10 μL+2490 μL PBS) to generate 40        μM pramipexole. 40 μM pramipexole is further diluted via a        100-fold dilution in 0.4% DMSO/PBS (100 μL+9900 μL Vehicle) to        generate 400 nM (4× assay concentration of the standard        pramipexole). 25 μL of 400 nM pramipexole is added to the        following wells of the Flashplate to yield 100 nM pramipexole        final; column 1 wells A-D and column 12 wells E-H.        Cells+forskolin are added later.        Cyclase Activation Flashplate Assay. (NEN SMP004B)    -   As described in the Materials section, forskolin is dissolved in        distilled water to achieve a stock concentration of 20 mM. This        is further diluted to 40 μM (4× assay concentration) using PBS.        25 μL of 40 μM stock is added to all wells using a Multi-drop,        giving a final concentration of 10 μM. Plates are then sealed        and incubated at 37° C. in a Westbart incubator while cells are        harvested.    -   Cells are harvested from flasks, which are between 70%-80%        confluent. It is essential that all components added to the        cells are warmed to 37° C. 5 mL of CDS is added per T225 flask        and incubated at 37° C. for 5 minutes before being neutralised        with 5 mL PBS. The cells are then centrifuged at 160 g (1000        rpm) for 5 minutes. The resultant supernatant is discarded and        cells are re-suspended in Stimulation Buffer (warmed to 37° C.),        to achieve 5×10⁵ cells/ml. 50 μl of cell suspension is then        dispensed into all wells of the Flashplate.    -   Plates are immediately incubated at 37° C. on a shaking        incubator for 15 minutes. The reaction is terminated with 100 μl        of Detection Mix in all wells (100 μL ¹²⁵I cAMP: 11 ml Detection        buffer per plate).    -   Plates are re-sealed and incubated in the dark for 3 hours to        allow equilibrium between the anti-cAMP antibody (coating the        wells), [¹²⁵I]-cAMP tracer and cellular cAMP.    -   Plates are counted on a Packard Topcount NXT using a suitable        ECADA compatible protocol (Protocol 75)

Resuscitation of Frozen Ampoules

Remove ampoules from liquid nitrogen and allow them to equilibrate for 2minutes as trapped gas or liquid may cause the ampoule to expand rapidlyand explode. They can also be placed at minus 20° C. for 2 minutesbefore thawing.

Thaw ampoules quickly and completely at 37° C. in a water bath.

Transfer cell suspension to a 75 cm² flask containing 10 mL growth mediaand incubate for 24 h at 37° C., 5% CO₂. After cell attachment (3-6hours) media is removed and replaced with fresh media (to remove DMSO).After 24 h, if approaching confluency, cells are transferred to a 225cm² flask. If not, the cells are maintained until they are 70%-80%confluent.

Cell Harvesting and Splitting

Cells are split on a Friday to provide cells for assays on Monday andTuesday. Cells required for the remainder of the week are split on aMonday.

It is essential not to allow the hD₃CHO cells grow beyond 80%confluence, or to create splits >1:20, as this has detrimental effectson their proliferative response and will subsequently effect the cellsability to perform in the assay.

Cells are grown in 225 cm² flasks (Jumbos). Every component added to thecells must be warmed to 37° C. before use.

Cell Harvest

Growth media is removed from flasks and cells are washed with warm PBS(Gibco. 14040-091) and removed.

-   -   5 mL of cell dissociation buffer is added to cells and placed in        incubator for approx. 5 minutes.    -   Flasks given a sharp tap to dislodge any remaining cells from        the tissue culture plastic.    -   5 mL of PBS is added to the cells and used to wash the base and        of the flask. Cells are centrifuged for 5 minutes at 160 g (1000        rpm) to pellet the cells.    -   Supernatant is discarded and 5 mL of Stimulation Buffer is used        to re-suspend the cells. A trypan blue exclusion assay is        carried out to determine the number of viable cells.    -   Cells are diluted in Stimulation Buffer to yield a concentration        of 5×10⁵ cells/ml.    -   To passage to cells the centrifugation step is omitted and the        cell suspension is dispensed into new T225 flasks containing 50        mL media.        Split Ratios

hD₃CHO are split between 1:5 to 1:10. The culture cannot be continuedbeyond passage

30 as cell line characteristics are lost with increased passage.

Cryopreservation of Cell Lines

It is essential to create a cell bank of your own cells to resuscitatefor further use.

-   -   Cells are harvested as described in the previous section.        Following the trypan blue exclusion assay, cells are diluted in        medium containing 10% DMSO to achieve 2 to 4×10⁶ cells/ml.    -   Cells are divided into 1 ml aliquots and immediately frozen down        gradually, in a ‘Mr Frosty’, (containing fresh IPA) at −80° C.        prior to being transferred to a gaseous-phase liquid-nitrogen        storage vessel. (Cells may be stored in the ‘Mr Frosty’ for up        to 2 days).

It is advisable to test the cell viability by thawing one ampoule afterfreezing. Viabilities below 70% may cause problems on recovery due tolow cell numbers and the presence of debris.

Data Analysis

The data is analysed using ECADA.

% Normalisation (in relation to pramipexole) is generated for allcompounds via the following formulae:% Normalisation=(X−B0)/(Max−B0)×100where,

-   x=Average net counts for a given concentration of test compound,-   Bo=Average net counts of minimum control (0 nM of Pramipexole) and,-   Max=Average net counts given of maximum control (100 nM Pramipexole)

Curves can be generated by plotting % normalisation (y) versusconcentration of agonist in nM (x). Data is fitted using non-linearregression with the slope constrained to 1. From this, an EC50 and %Emax for the test compound are determined.

Assay Plate Layout (10-point EC50):

Column 1: Wells A-D = MAX: High Controls (cells + forskolin + 100 nMpramipexole) Wells E-H = MIN: Low Controls (cells + forskolin + vehicle)Column 12: Wells A-D = MIN: Low Controls (cells + forskolin + vehicle)Wells E-H = MAX: High Controls (cells + forskolin + 100 nM pramipexole)Columns 2-11: 10-point serial dilutions (in duplicate) oftest-compounds. Decreasing concentrations from columns 2 to 11 (1000 nMto 0.03 nM). Pramipexole replaces C1 in first plate.Inhibition Via Dopamine D2 Receptors of Forskolin Stimulated AdenylateCyclase Activity

Materials

Cell Culture Media:

HD2 GH4C1/hD_(2L) M dium Hams F-12 (Sigma N6013) 2mM L-Glutamine (SigmaG7513) 10% FBS (Gibco 10106-169) 700 μg/ml Geneticin (Gibco 10131-019)

GH4C1/hD_(2L) are rat pituitary cells expressing the human dopamineD2_(long) receptor.

-   -   Media is made up fresh every week as below and filtered through        a 0.22 μM filter before use. Media is stored at 4° C. and warmed        to 37° C. for addition to the cells.        Cell Dissociation Solution (CDS): (Sigma C-5914)

5 mL used to harvest cells from 225 cm² flask

Phosphate Buffered Saline (PBS): (Gibco. 14040-091)

Trypan Blue: (Sigma T8154)

Forskolin (Calbiochem 344273)

Dissolved to a concentration of 20 mM in distilled water. (This stock isstored at +4° C.).

4× assay stock of 20 μM made by carrying out a 1000-fold dilution in PBSbuffer. 25 μl of the 20 μM stock is added to a final assay volume of 100μl, giving a final assay concentration of 5 μM.

Test Compounds

Dissolved to a concentration of 10 mM in 100% DMSO

Pramipexole Standard

Dissolved in 100% DMSO to yield a final stock concentration of 10 mM.

Cyclase Activation Flashplate Assay (NEN SMP004B)

Supplied by Perkin-Elmer Life Sciences, Inc

^([125I)]-cyclic Adenosine Monophosphate (cAMP) (NEX 130)

-   -   Supplied by Perkin-Elmer Life Sciences, Inc        Specific Equipment

Westbart Microtitre Plate Shaker/Incubator

Packard Topcount NXT (ECADA compatible programme)

Tecan Genesis

Labsystems Multi-drop DW

Protocols

Compound Dilutions

-   -   Pramipexole is included as a reference standard. A 10-point,        semi-log curve is generated every 4 plates. Compound responses        are normalised to the minimum (0 nM pramipexole) and maximum        (1000 nM Pramipexole) responses generated by the cells. All test        compounds may also be tested via a 10-point (semi-log) curve.    -   Test compounds are dissolved in 100% DMSO to yield a stock        concentration of 10 mM, (1000× the final assay concentration        required, e.g. 10 mM will give a top concentration of 10000 nM).    -   Pramipexole is dissolved in 100% DMSO to give a concentration of        10 mM. Pramipexole is diluted further to 1 mM in 100% DMSO via a        10-fold dilution.    -   Further dilutions and additions are carried out in 0.4% DMSO/PBS        using a suitable Tecan Genesis Protocol which is capable of        performing serial dilutions at a fold of 3.159 (semi-log unit).

Tecan Genesis Dilutions

-   -   10 μL of the test compounds are added to column 1 of a        microplate. 240 μL of 0.4% DMSO/PBS is added to this to give a        25-fold dilution (0.4 mM). 20 μL of the 0.4 mM dilution is        transferred to wells of column 2 where 180 μL of 0.4% DMSO/PBS        is added, giving a further 10-fold dilution to achieve a 4× top        assay concentration (0.04 mM).    -   Serial dilutions are performed (3.159-fold) to achieve a        semi-log dilution series:        -   40 μM, 12.7 μM, 4 μM, 1.27 μM, 400 nM, 130 nM, 40 nM, 13 nM,            4 nM, 1.3 nM    -   25 μL (in duplicate) of the serial dilutions are transferred to        columns 2-11 of the Flashplate (See Appendix). Since the finals        assay volume is 100 μL, the final assay concentrations will be:        -   10,000 uM, 3170 nM, 1000 nM, 320 nM, 100 nM, 32 nM, 10 nM, 3            nM, 1 nM, 0.3 nM    -   Minimum control (low control): 25 μL of 0.4% DMSO/PBS (vehicle)        is added to the following wells (column 1 wells E-H and column 2        wells A-D). Cells and forskolin are added later.    -   Maximum control (high control): 10 mM pramipexole is diluted in        PBS via a 250-fold dilution (10 μL+2490 μL PBS) to generate 40        μM pramipexole. 40 μM pramipexole is further diluted via a        10-fold dilution in 0.4% DMSO/PBS (100 μL+990 μL Vehicle) to        generate 4000 nM (4× assay concentration of the standard        pramipexole). 25 μL of 4000 nM pramipexole is added to the        following wells of the Flashplate to yield 1000 nM pramipexole        final; column 1 wells A-D and column 12 wells E-H.        Cells+forskolin are added later.        Cyclase-Activation Flashplate Assay. (NEN SMP004B)    -   As described in the Materials section, forskolin is dissolved in        distilled water to achieve a stock concentration of 20 mM. This        is further diluted to 20 μM (4× assay concentration) using PBS.        25 μL is added to all wells using a Multi-drop, giving a final        concentration of 5 μM. Plates are then sealed and incubated at        37° C. in a Westbart incubator while cells are harvested.    -   Cells are harvested from flasks which are between 70%-80%        confluent. It is essential that all components added to the        cells are warmed to 37° C. 5 mL of CDS is added per 225 cm²        flask, and incubated at 37° C. for 5 minutes before being        neutralised with 5 mL PBS. The cells are then centrifuged at 160        g (1000 rpm) for 5 minutes. The resultant supernatant is        discarded and cells are re-suspended in Stimulation Buffer        (warmed to 37° C.), to achieve 1×10⁵ cells/ml. 50 μl of cell        suspension is then dispensed into all wells of the Flashplate.    -   Plates are immediately incubated at 37° C. on a shaking        incubator for 15 minutes. The reaction is terminated with 100 μl        of Detection Mix in all wells (100 μL ¹²⁵I cAMP: 11 ml Detection        buffer plate).    -   Plates are re-sealed and incubated in the dark for 3 hours to        allow equilibrium between the anti-cAMP antibody (coating the        wells), [¹²⁵I]-cAMP tracer and cellular cAMP.    -   Plates are counted on a Packard Topcount NXT using a suitable        ECADA compatible protocol (Protocol 75)

Resuscitation of Frozen Ampoules

Remove ampoules from liquid nitrogen and allow them to equilibrate for 2minutes as trapped gas or liquid may cause the ampoule to expand rapidlyand explode. They can also be placed at minus 20° C. for 2 minutesbefore thawing.

Thaw ampoules quickly and completely at 37° C. in a water bath.

Transfer cell suspension to a 75 cm² flask containing 10 mL growth mediaand incubate for 24 h at 37° C., 5% CO₂. After cell attachment (3-6hours) media is removed and replaced with fresh media (to remove DMSO).After 24 h, if approaching confluency, cells are transferred to a 225cm² flask. If not, the cells are maintained until they are 60%confluent.

Cell Harvesting and Splitting

Cells are split on a Friday to provide cells for assays on Monday andTuesday. Cells required for the remainder of the week are split on aMonday.

It is essential not to allow the cells grow beyond 60% confluence asthis has detrimental effects on their proliferative response and willsubsequently effect the cells ability to perform in the assay.

Cells are grown in 225 cm² flasks (Jumbos). Every component added to thecells must be warmed to 37° C. before use.

Cell harv st

Growth media is removed from flasks and cells are washed with warm PBS(Gibco. 14040-091) and removed.

-   -   5 mL of cell dissociation buffer is added to cells and placed in        incubator for approx. 5 minutes.    -   Flasks given a sharp tap to dislodge any remaining cells from        the tissue culture plastic.    -   5 mLof PBS is added to the cells and used to wash the base and        of the flask. Cells are centrifuged for 5 minutes at 160 g (1000        rpm) to pellet the cells.    -   Supernatant is discarded and 5 mL of Stimulation Buffer is used        to re-suspend the cells. A trypan blue exclusion assay is        carried out to determine the number of viable cells.    -   Cells are diluted in Stimulation Buffer to yield a concentration        of 1×10⁵ cells/ml.    -   To passage to cells the centrifugation step is omitted and the        cell suspension is dispensed into new T225 flasks containing 50        mL media.        Split Ratios

GH4C1/D2 are split between 1:3 to 1:5.

Cryopreservation of Cell Lines

It is essential to create a cell bank of your own cells to resuscitatefor further use.

-   -   Cells are harvested as described in the previous section.        Following the trypan blue exclusion assay, cells are diluted in        medium containing 10% DMSO to achieve 2 to 4×10⁶ cells/ml.    -   Cells are divided into 1 ml aliquots and immediately frozen down        gradually, in a ‘Mr Frosty’, (containing fresh IPA) at −80° C.        prior to being transferred to a gaseous-phase liquid-nitrogen        storage vessel. (Cells may be stored in the ‘Mr Frosty’ for up        to 2 days).

It is advisable to test the cell viability by thawing one ampoule afterfreezing. Viabilities below 70% may cause problems on recovery due tolow cell numbers and the presence of debris.

Data Analysis

The data is analysed using ECADA.

% Normalisation (in relation to pramipexole) is generated for allcompounds via the following formulae:% Normalisation=(X−B0)/(Max−B0)×100where,

-   x=Average net counts for a given concentration of test compound,-   Bo=Average net counts of minimum control (0 nM of Pramipexole) and,-   Max=Average net counts given of maximum control (100 nM Pramipexole)

Curves can be generated by plotting % normalisation (y) versusconcentration of agonist in nM (x). Data is fitted using non-linearregression with the slope constrained to 1. From this, an EC50 and %Emax for the test compound are determined.

Assay Plate Layout (10-point EC50):

Column 1: Wells A-D = MAX: High Controls (cells + forskolin + 100 nMpramipexole) Wells E-H = MIN: Low Controls (cells + forskolin + vehicle)Column 12: Wells A-D = MIN: Low Controls (cells + forskolin + vehicle)Wells E-H = MAX: High Controls (cells + forskolin + 100 nM pramipexole)Columns 2-11: 10-point serial dilutions (in duplicate) oftest-compounds. Decreasing concentrations from columns 2 to 11 (1000 nMto 0.03 nM). Pramipexole replaces C1 in first plate.

Using the assay described above, the compounds of the present inventionall exhibit a functional potency at D3 receptor expressed as an EC50,lower than 1000 nM and a 10 fold selectivity for D3 over D2.

Compound of example 8 has a functional potency at D3 receptor expressedas an EC50, of 7.6 nM and 1315.8 fold selectivity for D3 over D2.Selectivity is calculated as the D2 EC50 value divided by the D3 EC50value. Where the value of the D2 EC50 was >10000, a figure of 10000 wasused in the calculation.

It is to be appreciated that all references herein to treatment includecurative, palliative and prophylactic treatment.

Suitable auxiliary active agents for use in the combinations of thepresent invention include:

-   1) Naturally occurring or synthetic prostaglandins or esters    thereof. Suitable prostaglandins for use herein include compounds    such as alprostadil, prostaglandin E₁, prostaglandin E₀,    13,14-dihydroprosta glandin E₁, prostaglandin E₂, eprostinol,    natural synthetic and semi-synthetic prostaglandins and derivatives    thereof including those described in WO-00033825 and/or U.S. Pat.    No. 6,037,346 issued on 14 Mar. 2000 all incorporated herein by    reference, PGE₀, PGE₁, PGA₁, PGB₁, PGF₁ α, 19-hydroxy PGA₁,    19-hydroxy-PGB₁, PGE₂, PGB₂, 19-hydroxy-PGA₂, 19-hydroxy-PGB₂,    PGE₃α, carboprost tromethamine dinoprost, tromethamine,    dinoprostone, lipo prost, gemeprost, metenoprost, sulprostune,    tiaprost and moxisylate;-   2) α-adrenergic receptor antagonist compounds also known as    α-adrenoceptors or α-receptors or α-blockers. Suitable compounds for    use herein include: the α-adrenergic receptor blockers as described    in PCT application WO99/30697 published on 14 Jun. 1998, the    disclosures of which relating to α-adrenergic receptors are    incorporated herein by reference and include, selective    α₁-adrenoceptor or α₂-adrenoceptor blockers and non-selective    adrenoceptor blockers, suitable α₁-adrenoceptor blockers include:    phentolamine, phentolamine mesylate, trazodone, alfuzosin,    indoramin, naftopidil, tamsulosin, dapiprazole, phenoxybenzamine,    idazoxan, efaraxan, yohimbine, rauwolfa alkaloids, Recordati    15/2739, SNAP 1069, SNAP 5089, RS17053, SL 89.0591, doxazosin,    terazosin, abanoquil and prazosin; α₂-blocker blockers from U.S.    Pat. No. 6,037,346 [14th Mar. 2000] dibenarnine, tolazoline,    trimazosin and dibenarnine; α-adrenergic receptors as described in    U.S. Pat. Nos.: 4,188,390; 4,026,894; 3,511,836; 4,315,007;    3,527,761; 3,997,666; 2,503,059; 4,703,063; 3,381,009; 4,252,721 and    2,599,000 each of which is incorporated herein by reference;    α₂-Adrenoceptor blockers include: clonidine, papaverine, papaverine    hydrochloride, optionally in the presence of a cariotonic agent such    as pirxamine;-   3) NO-donor (NO-agonist) compounds. Suitable NO-donor compounds for    use herein include organic nitrates, such as mono- di or    tri-nitrates or organic nitrate esters including glyceryl trinitrate    (also known as nitroglycerin), isosorbide 5-mononitrate, isosorbide    dinitrate, pentaerythritol tetranitrate, erythrityl tetranitrate,    sodium nitroprusside (SNP), 3-morpholinosydnonimine molsidomine,    S-nitroso-N-acetyl penicilliamine (SNAP) S-nitroso-N-glutathione    (SNO-GLU), N-hydroxy-L-arginine, amylnitrate, linsidomine,    linsidomine chlorohydrate, (SIN-1) S-nitroso-N-cysteine, diazenium    diolates, (NONOates), 1,5-pentanedinitrate, L-arginene, ginseng,    zizphi fructus, molsidomine, Re-2047, nitrosylated maxisylyte    derivatives such as NMI-678-11 and NMI-937 as described in published    PCT application WO 0012075;-   4) Potassium channel openers or modulators. Suitable potassium    channel openers/modulators for use herein include nicorandil,    cromokalim, levcromakalim, lemakalim, pinacidil, cliazoxide,    minoxidil, charybdotoxin, glyburide, 4-amini pyridine, BaCl₂;-   5) Vasodilator agents. Suitable vasodilator agents for use herein    include nimodepine, pinacidil, cyclandelate, isoxsuprine,    chloroprumazine, halo peridol, Rec 15/2739, trazodone;-   6) Thromboxane A2 agonists;-   7) CNS active agents;-   8) Ergot alkoloids; Suitable ergot alkaloids are described in U.S.    Pat. No. 6,037,346 issued on 14 Mar. 2000 and include acetergamine,    brazergoline, bromerguride, cianergoline, delorgotrile, disulergine,    ergonovine maleate, ergotamine tartrate, etisulergine, lergotrile,    lysergide, mesulergine, metergoline, metergotamine, nicergoline,    pergolide, propisergide, proterguride and terguride;-   9) Compounds which modulate the action of natruretic factors in    particular atrial naturetic factor (also known as atrial naturetic    peptide), B type and C type naturetic factors such as inhibitors or    neutral endopeptidase;-   10) Compounds which inhibit angiotensin-converting enzyme such as    enapril, and combined inhibitors of angiotensin-converting enzyme    and neutral endopeptidase such as omapatrilat.-   11) Angiotensin receptor antagonists such as losartan;-   12) Substrates for NO-synthase, such as L-arginine;-   13) Calcium channel blockers such as amlodipine;-   14) Antagonists of endothelin receptors and inhibitors or    endothelin-converting enzyme;-   15) Cholesterol lowering agents such as statins (e.g.    atorvastatin/Lipitor-trade mark) and fibrates;-   16) Antiplatelet and antithrombotic agents, e.g. tPA, uPA, warfarin,    hirudin and other thrombin inhibitors, heparin, thromboplastin    activating factor inhibitors;-   17) Insulin sensitising agents such as rezulin and hypoglycaemic    agents such as glipizide;-   18) L-DOPA or carbidopa;-   19) Acetylcholinesterase inhibitors such as donezipil;-   20) Steroidal or non-steroidal anti-inflammatory agents;-   21) Estrogen receptor modulators and/or estrogen agonists and/or    estrogen antagonists, preferably raloxifene or lasofoxifene,    (−)-cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol    and pharmaceutically acceptable salts thereof the preparation of    which is detailed in WO 96/21656;-   23) A PDE inhibitor, more particularly a PDE 2, 3, 4, 5, 7 or 8    inhibitor, preferably PDE2 or PDE5 inhibitor and most preferably a    PDE5 inhibitor (see hereinafter), said inhibitors preferably having    an IC50 against the respective enzyme of less than 10nM (with the    proviso that PDE 3 and 4 inhibitors are only administered topically    or by injection to the penis);-   22) Vasoactive intestinal protein (VIP), VIP mimetic, VIP analogue,    more particularly mediated by one or more of the VIP receptor    subtypes VPAC1, VPAC or PACAP (pituitory adenylate cyclase    activating peptide), one or more of a VIP receptor agonist or a VIP    analogue (e.g. Ro-125-1553) or a VIP fragment, one or more of a    α-adrenoceptor antagonist with VIP combination (e.g. Invicorp,    Aviptadil);-   23) A melanocortin receptor agonist or modulator or melanocortin    enhance, such as melanotan II, PT-14, PT-141 or compounds claimed in    WO-09964002, WO-00074679, WO-09955679, WO-00105401, WO-00058361,    WO-00114879, WO-00113112, WO-09954358;-   24) A serotonin receptor agonist, antagonist or modulator, more    particularly agonists, antagonists or modulators for 5HT1A    (including VML 670), 5HT2A, 5HT2C, 5HT3 and/or 5HT6 receptors,    including those described in WO-09902159, WO-00002550 and/or    WO-00028993;-   25) A testosterone replacement agent (including    dehydroandrostendione), testosternone (Tostrelle),    dihydrotestosterone or a testosterone implant;-   26) Estrogen, estrogen and medroxyprogesterone or    medroxyprogesterone acetate (MPA) (i.e. as a combination), or    estrogen and methyl testosterone hormone replacement therapy agent    (e.g. HRT especially Premarin, Cenestin, Oestrofeminal, Equin,    Estrace, Estrofem, Elleste Solo, Estring, Eastraderm TTS, Eastraderm    Matrix, Dermestril, Premphase, Preempro, Prempak, Premique,    Estratest, Estratest HS, Tibolone);-   27) A modulator of transporters for noradrenaline, dopamine and/or    serotonin, such as bupropion, GW-320659;-   28) A purinergic receptor agonist and/or modulator;-   29) A neurokinin (NK) receptor antagonist, including those described    in WO-09964008;-   30) An opioid receptor agonist, antagonist or modulator, preferably    agonists for the ORL-1 receptor;-   31) An agonist or modulator for oxytocin/vasopressin receptors,    preferably a selective oxytocin agonist or modulator;-   32) Modulators of cannabinoid receptors;-   33) A SEP inhibitor (SEPi), for instance a SEPi having an IC₅₀ at    less than 100 nanomolar, more preferably, at less than 50 nanomolar.    Preferably, the SEP inhibitors according to the present invention    have greater than 30-fold, more preferably greater than 50-fold    selectivity for SEP over neutral endopeptidase NEP EC 3.4.24.11 and    angiotensin converting enzyme (ACE). Preferably the SEPi also has a    greater than 100-fold selectivity over endothelin converting enzyme    (ECE).

By cross reference herein to compounds contained in patents and patentapplications which can be used in accordance with invention, we mean thetherapeutically active compounds as defined in the claims (in particularof claim 1) and the specific examples (all of which is incorporatedherein by reference).

If a combination of active agents is administered, then they may beadministered simultaneously, separately or sequentially.

Auxiliary Agents—PDE5 Inhibitors

The suitability of any particular cGMP PDE5 inhibitor can be readilydetermined by evaluation of its potency and selectivity using literaturemethods followed by evaluation of its toxicity, absorption, metabolism,pharmacokinetics, etc in accordance with standard pharmaceuticalpractice.

IC50 values for the cGMP PDE5 inhibitors may be determined using thePDE5 assay (see hereinbelow).

Preferably the cGMP PDE5 inhibitors used in the pharmaceuticalcombinations according to the present invention are selective for thePDE5 enzyme. Preferably (when used orally) they are selective over PDE3,more preferably over PDE3 and PDE4. Preferably (when oral), the cGMPPDE5 inhibitors of the invention have a selectivity ratio greater than100 more preferably greater than 300, over PDE3 and more preferably overPDE3 and PDE4.

Selectivity ratios may readily be determined by the skilled person. IC50values for the PDE3 and PDE4 enzyme may be determined using establishedliterature methodology, see S A Ballard et al, Journal of Urology, 1998,vol. 159, pages 2164-2171 and as detailed herein after.

Suitable CGMP PDE5 inhibitors for the use according to the presentinvention include:

-   -   the pyrazolo [4,3-d]pyrimidin-7-ones disclosed in EP-A-0463756;        the pyrazolo [4,3-d]pyrimidin-7-ones disclosed in EP-A-0526004;        the pyrazolo [4,3-d]pyrimidin-7-ones disclosed in published        international patent application WO 93/06104; the isomeric        pyrazolo [3,4-d]pyrimidin-4-ones disclosed in published        international patent application WO 93/07149; the        quinazolin-4-ones disclosed in published international patent        application WO 93/12095; the pyrido [3,2-d]pyrimidin-4-ones        disclosed in published international patent application WO        94/05661; the purin-6-ones disclosed in published international        patent application WO 94/00453; the pyrazolo        [4,3-d]pyrimidin-7-ones disclosed in published international        patent application WO 98/49166; the pyrazolo        [4,3-d]pyrimidin-7-ones disclosed in published international        patent application WO 99/54333; the pyrazolo        [4,3-d]pyrimidin-4-ones disclosed in EP-A-0995751; the pyrazolo        [4,3-d]pyrimidin-7-ones disclosed in published international        patent application WO 00/24745; the pyrazolo        [4,3-d]pyrimidin-4-ones disclosed in EP-A-0995750; the compounds        disclosed in published international application WO95/19978; the        compounds disclosed in published international application WO        99/24433 and the compounds disclosed in published international        application WO 93/07124. The pyrazolo [4,3-d]pyrimidin-7-ones        disclosed in published international application WO 01/27112;        the pyrazolo [4,3-d]pyrimidin-7-ones disclosed in published        international application WO 01/27113; the compounds disclosed        in EP-A-1092718 and the compounds disclosed in EP-A-1092719.

Further suitable PDE5 inhibitors for the use according to the presentinvention include:

-   -   5-[2-ethoxy-5-(4-methyl-1-piperazinylsulphonyl)phenyl]-1-methyl-3-n-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one        (sildenafil) also known as        1-[[3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-5-yl)-4-ethoxyphenyl]sulphonyl]-4-methylpiperazine        (see EP-A-0463756);        5-(2-ethoxy-5-morpholinoacetylphenyl)-1-methyl-3-n-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one        (see EP-A-0526004);        3-ethyl-5-[5-(4-ethylpiperazin-1-ylsulphonyl)-2-n-propoxyphenyl]-2-(pyridin-2-yl)methyl-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one        (see WO98/49166);        3-ethyl-5-[5-(4-ethylpiperazin-1-ylsulphonyl)-2-(2-methoxyethoxy)pyridin-3-yl]-2-(pyridin-2-yl)methyl-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one        (see WO99/54333);        (+)-3-ethyl-5-[5-(4-ethylpiperazin-1-ylsulphonyl)-2-(2-methoxy-1(R)-methylethoxy)pyridin-3-yl]-2-methyl-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one,        also known as        3-ethyl-5-{5-[4-ethylpiperazin-1-ylsulphonyl]-2-([(1R)-2-methoxy-1-methylethyl]oxy)pyridin-3-yl}-2-methyl-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one        (see WO99/54333);        5-[2-ethoxy-5-(4-ethylpiperazin-1-ylsulphonyl)pyridin-3-yl]-3-ethyl-2-[2-methoxyethyl]-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one,        also known as        1-{6-ethoxy-5-[3-ethyl-6,7-dihydro-2-(2-methoxyethyl)-7-oxo-2H-pyrazolo[4,3-d]pyrimidin-5-yl]-3-pyridylsulphonyl}-4-ethylpiperazine        (see WO 01/27113, Example 8);        5-[2-iso-Butoxy-5-(4-ethylpiperazin-1-ylsulphonyl)pyridin-3-yl]-3-ethyl-2-(1-methylpiperidin-4-yl)-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one        (see WO 01/27113, Example 15);        5-[2-Ethoxy-5-(4-ethylpiperazin-1-ylsulphonyl)pyridin-3-yl]-3-ethyl-2-phenyl-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one        (see WO 01/27113, Example 66);        5-(5-Acetyl-2-propoxy-3-pyridinyl)-3-ethyl-2-(1-isopropyl-3-azetidinyl)-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one        (see WO 01/27112, Example 124);        5-(5-Acetyl-2-butoxy-3-pyridinyl)-3-ethyl-2-(1-ethyl-3-azetidinyl)-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one        (see WO 01/27112, Example 132);        (6R,12aR)-2,3,6,7,12,12a-hexahydro-2-methyl-6-(3,4-methylenedioxyphenyl)-pyrazino[2′,1′:6,1]pyrido[3,4-b]indole-1,4-dione        (IC-351), i.e. the compound of examples 78 and 95 of published        international application WO95/19978, as well as the compound of        examples 1, 3, 7 and 8;        2-[2-ethoxy-5-(4-ethyl-piperazin-1-yl-1-sulphonyl)-phenyl]-5-methyl-7-propyl-3H-imidazo[5,1-f][1,2,4]triazin-4-one        (vardenafil) also known as        1-[[3-(3,4-dihydro-5-methyl-4-oxo-7-propylimidazo[5,1-f]-as-triazin-2-yl)-4-ethoxyphenyl]sulphonyl]-4-ethylpiperazine,        i.e. the compound of examples 20, 19, 337 and 336 of published        international application WO99/24433; and the compound of        example 11 of published international application WO93/07124        (EISAl); and compounds 3 and 14 from Rotella D P, J. Med. Chem.,        2000, 43, 1257.

Still other suitable PDE5 inhibitors include:

-   -   4-bromo-5-(pyridylmethylamino)-6-[3-(4-chlorophenyl)-propoxy]-3(2H)pyridazinone;        1-[4-[(1,3-benzodioxol-5-ylmethyl)amiono]-6-chloro-2-quinozolinyl]-4-piperidine-carboxylic        acid, monosodium salt;        (+)-cis-5,6a,7,9,9,9a-hexahydro-2-[4-(trifluoromethyl)-phenylmethyl-5-methylcyclopent-4,5]imidazo[2,1-b]purin-4(3H)one;        furaziocillin;        cis-2-hexyl-5-methyl-3,4,5,6a,7,8,9,9a-octahydrocyclopent[4,5]-imidazo[2,1-b]purin-4-one;        3-acetyl-1-(2-chlorobenzyl)-2-propylindole-6-carboxylate;        3-acetyl-1-(2-chlorobenzyl)-2-propylindole-6-carboxylate;        4-bromo-5-(3-pyridylmethylamino)-6-(3-(4-chlorophenyl)        propoxy)-3-(2H)pyridazinone;        l-methyl-5(5-morpholinoacetyl-2-n-propoxyphenyl)-3-n-propyl-1,6-dihydro-7H-pyrazolo(4,3-d)pyrimidin-7-one;        1-[4-[(1,3-benzodioxol-5-ylmethyl)arnino]-6-chloro-2-quinazolinyl]-4-piperidinecarboxylic        acid, monosodium salt; Pharmaprojects No. 4516 (Glaxo Wellcome);        Pharmaprojects No. 5051 (Bayer); Pharmaprojects No. 5064 (Kyowa        Hakko; see WO 96/26940); Pharmaprojects No. 5069 (Schering        Plough); GF-196960 (Glaxo Wellcome); E-8010 and E-4010 (Eisai);        Bay-38-3045 & 38-9456 (Bayer) and Sch-51866.

The compounds of the formula (I) can be administered alone but willgenerally be administered in admixture with a suitable pharmaceuticalexcipient, diluent or carrier selected with regard to the intended routeof administration and standard pharmaceutical practice.

Accordingly the present invention provides for a composition comprisinga compound of formula (I), (Ia) or (Ib) and a pharmaceuticallyacceptable diluent or carrier.

For example, the compounds of the formula (I), (Ia) or (Ib) can beadministered orally, buccally or sublingually in the form of tablets,capsules, ovules, elixirs, solutions or suspensions, which may containflavouring or colouring agents, for immediate-, delayed-, modified-,sustained-, pulsed- or controlled-release applications.

Such tablets may contain excipients such as microcrystalline cellulose,lactose, sodium citrate, calcium carbonate, dibasic calcium phosphateand glycine, disintegrants such as starch (preferably corn, potato ortapioca starch), sodium starch glycollate, croscarmellose sodium andcertain complex silicates, and granulation binders such aspolyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC),hydroxypropylcellulose (HPC), sucrose, gelatin and acacia. Additionally,lubricating agents such as magnesium stearate, stearic acid, glycerylbehenate and talc may be included.

Solid compositions of a similar type may also be employed as fillers ingelatin capsules. Preferred excipients in this regard include lactose,starch, a cellulose, milk sugar or high molecular weight polyethyleneglycols. For aqueous suspensions and/or elixirs, the compounds of theformula (I), (Ia) or (Ib) may be combined with various sweetening orflavouring agents, colouring matter or dyes, with emulsifying and/orsuspending agents and with diluents such as water, ethanol, propyleneglycol and glycerin, and combinations thereof.

The compounds of the formula (I), (Ia) or (Ib) can also be administeredparenterally, for example, intravenously, intra-arterially,intraperitoneally, intrathecally, intraventricularly, intraurethrally,intrasternally, intracranially, intramuscularly or subcutaneously, orthey may be administered by infusion techniques. For such parenteraladministration they are best used in the form of a sterile aqueoussolution which may contain other substances, for example, enough saltsor glucose to make the solution isotonic with blood. The aqueoussolutions should be suitably buffered (preferably to a pH of from 3 to9), if necessary. The preparation of suitable parenteral formulationsunder sterile conditions is readily accomplished by standardpharmaceutical techniques well-known to those skilled in the art.

The compounds of formula (I), (Ia) or (Ib) can also be administeredintranasally or by inhalation, typically in the form of a dry powder(either alone, as a mixture, for example, in a dry blend with lactose,or as a mixed component particle, for example, mixed with phospholipids)from a dry powder inhaler or as an aerosol spray from a pressurisedcontainer, pump, spray, atomiser (preferably an atomiser usingelectrohydrodynamics to produce a fine mist), or nebuliser, with orwithout the use of a suitable propellant, such as dichlorofluoromethane.

The pressurised container, pump, spray, atomizer, or nebuliser containsa solution or suspension of the active compound comprising, for example,ethanol (optionally, aqueous ethanol) or a suitable alternative agentfor dispersing, solubilising, or extending release of the active, thepropellant(s) as_solvent and an optional surfactant, such as sorbitantrioleate or an oligolactic acid.

Prior to use in a dry powder or suspension formulation, the drug productis micronised to a size suitable for delivery by inhalation (typicallyless than 5 microns). This may be achieved by any appropriatecomminuting method, such as spiral jet milling, fluid bed jet milling,supercritical fluid processing to form nanoparticles, high pressurehomogenisation, or spray drying.

A suitable solution formulation for use in an atomiser usingelectrohydrodynamics to produce a fine mist may contain from 1 μg to 10mg of the compound of the invention per actuation and the actuationvolume may vary from 1 μl to 100 μl. A typical formulation may comprisea compound of formula (I), (Ia) or (Ib), propylene glycol, sterilewater, ethanol and sodium chloride. Alternative solvents which may beused instead of propylene glycol include glycerol and polyethyleneglycol.

Capsules, blisters and cartridges (made, for example, from gelatin orHPMC) for use in an inhaler or insufflator may be formulated to containa powder mix of the compound of the invention, a suitable powder basesuch as lactose or starch and a performance modifier such as l-leucine,mannitol, or magnesium stearate.

Formulations for inhaled/intranasal administration may be formulated tobe immediate and/or modified release.

Alternatively, the compounds of the formula (I), (Ia) or (Ib) can beadministered in the form of a suppository or pessary, or they may beapplied topically in the form of a gel, hydrogel, lotion, solution,cream, ointment or dusting powder. The compounds of the formula (I),(Ia) or (Ib) may also be dermally or transdermally administered, forexample, by the use of a skin patch. They may also be administered bythe pulmonary or rectal routes.

They may also be administered by the ocular route. For ophthalmic use,the compounds can be formulated as micronised suspensions in isotonic,pH adjusted, sterile saline, or, preferably, as solutions in isotonic,pH adjusted, sterile saline, optionally in combination with apreservative such as a benzylalkonium chloride. Alternatively, they maybe formulated in an ointment such as petrolatum.

For application topically to the skin, the compounds of the formula (I),(Ia) or (Ib) can be formulated as a suitable ointment containing theactive compound suspended or dissolved in, for example, a mixture withone or more of the following: mineral oil, liquid petrolatum, whitepetrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound,emulsifying wax and water. Alternatively, they can be formulated as asuitable lotion or cream, suspended

or dissolved in, for example, a mixture of one or more of the following:mineral oil, sorbitan monostearate, a polyethylene glycol, liquidparaffin, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water.

The compounds of the formula (I), (Ia) or (Ib) may also be used incombination with a cyclodextrin. Cyclodextrins are known to forminclusion and non-inclusion complexes with drug molecules. Formation ofa drug-cyclodextrin complex may modify the solubility, dissolution rate,bioavailability and/or stability property of a drug molecule.Drug-cyclodextrin complexes are generally useful for most dosage formsand administration routes. As an alternative to direct complexation withthe drug the cyclodextrin may be used as an auxiliary additive, e.g. asa carrier, diluent or solubiliser. Alpha-, beta- and gamma-cyclodextrinsare most commonly used and suitable examples are described inWO-A-91/11172, WO-A-94/02518 and WO-A-98/55148.

The present invention is further exemplified by the following,non-limiting examples:

The invention is illustrated by the following non-limiting examples inwhich the following abbreviations and definitions are used:

α_(D) optical rotation at 587 nm. Arbacel ® filter agent b broad Boctert-butoxycarbonyl CDCl₃ chloroform-d1 CD₃OD methanol-d4 δ chemicalshift d doublet dd double doublet DCM dichloromethane DMFN,N-dimethylformamide DMSO dimethylsulfoxide h hours HCl hydrogenchloride LRMS low resolution mass spectrum m multiplet m/z mass spectrumpeak min minutes Mpt melting point NaOH sodium hydroxide NMR nuclearmagnetic resonance q quartet s singlet t triplet Tftrifluoromethanesulfonyl TFA trifluoroacetic acid THF tetrahydrofuranTLC thin layer chromatographyMelting points were determined using a Perkin Elmer DSC7 at a heatingrate of 20° C./minute).

X-RAY DIFFRACTION DATA WERE RECORDED AT ROOM TEMPERATURE USING A BRUKERAXS SMART-APEX CCD AREA-DETECTOR DIFFRACTOMETER (MO Kα RADIATION).INTENSITIES WERE INTEGRATED FROM SEVERAL SERIES OF EXPOSURES. EACHEXPOSURE COVERED 0.3° IN ω, WITH AN EXPOSURE TIME OF 60 S AND THE TOTALDATA SET WAS MORE THAN A SPHERE.

EXAMPLE 1 2-Amino-1-(3-methoxyphenyl)ethanol

3-Methoxybenzaldehyde (27.2 g, 0.2 mol) in THF (150 ml) was added to astirred solution of 3N HCl (aq) (150 ml, 0.3 mol) and sodium sulphite(37.8 g, 0.3 mol) at room temperature. After 10 minutes potassiumcyanide (19.53 g, 0.3 mol) was added, portion wise, and the reactionmixture was then stirred for 30 minutes. Diethyl ether (800 ml) andwater (300 ml) were added and subsequent layers partitioned. Aqueousre-extracted with diethyl ether (500 ml) the organics combined, driedover anhydrous magnesium sulphate, filtered then concentrated in vacuoto give the cyanohydrin intermediate as a colourless oil, (35.57 g, 0.22mol, >100%). Borane-tetrahydrofuran complex (1M in THF) (400 ml, 0.4mol) was then cautiously added to the cyanohydrin in THF (100 ml.). Onceeffervescence had ceased, stirring was continued at reflux for 1.5 hoursunder an atmosphere of nitrogen. The reaction mixture was cooled thenquenched with methanol (40 ml) before concentrating in vacuo to give acolourless oil. 6M HCl (aq) (200 ml) was added and reaction stirred atreflux for two hours before concentrating in vacuo to give a whitesolid. This was pre-absorbed onto silica then purified by columnchromatography eluting with dichloromethane: methanol: ammonia (90:10:1)to give the title compound as a colourless oil (31.3 g, 0.19 mol, 94%).¹H NMR (CDCl₃, 400 MHz) δ: 1.60 (bs, 2H), 2.80 (dd, 1H), 3.02 (dd, 1H),3.46 (s, 1H), 3.81 (s, 3H), 4.60 (dd, 1H), 6.81 (d, 1H), 6.91 (d, 1H),6.93 (s, 1H), 7.22 (t, 1H). LRMS: m/z 168 (M−H⁺). Analysis found C,56.66; H, 8.28; N, 6.91%. C₉H₁₃NO₂.1.33H₂O requires C, 56.33; H, 8.27;N, 7.30%.

EXAMPLE2 N-[2-Hydroxy-2-(3-methoxyphenyl)ethyl]propionamide

Triethylamine (52 ml, 0.37 mol) was added to the amine from example 1(31.3 g, 0.19 mol) in dichloromethane (400 ml) and reaction mixturestirred under an atmosphere of nitrogen gas at 0° C. for 10 minutes.Propionyl chloride (16.3 ml, 0.19 mol) was added and after stirring for30 minutes, the reaction temperature was raised to room temperature fora further 5 hours. The reaction mixture was quenched 1 N HCl (aq) (100ml) and then extracted with dichloromethane (2×50 ml). The organicfractions were combined, dried over anhydrous magnesium sulphate,filtered and concentrated in vacuo to give the title compound as acolourless oil that crystallised on standing to white crystals (28 g,0.13 mol, 67%). ¹H NMR (CDCl₃, 400 MHz) δ: 1.18 (t, 3H), 2.22 (q, 2H),2.51 (bs, 1H), 3.31 (m, 1H), 3.71 (dd, 1H), 3.80 (s, 3H), 4.81 (m, 1H),5.95 (bs, 1H), 6.80 (d, 1H), 6.90 (d, 1H), 6.91 (s, 1H), 7.22 (t, 1H).LRMS: m/z 224. Mpt: 77-78° C. Analysis found C, 63.86; H, 7.82; N,6.28%. C₁₂H₁₇NO₃.0.1H₂O requires C, 64.04; H, 7.70; N, 6.22%.

EXAMPLE 3 1-(3-Methoxyphenyl)-2-propylaminoethanol

Borane-tetrahydrofuran complex (1 M in THF) (376 ml, 0.4 mol) was addedto amide from example 2 (28 g, 0.13 mol) in dry THF (100 ml) then thereaction mixture, stirred under an atmosphere of nitrogen gas, wasbrought to reflux for 2.5 hours. The reaction mixture was cooled thenquenched with methanol (40 ml), before concentrating in vacuo to give anopaque white oil. 6N HCl (aq) (200 ml) was added and reaction stirred atreflux for two hours. The reaction mixture was cooled thendichloromethane (200 ml) added and the layers separated. The aqueouslayer was rendered basic by addition of potassium carbonate thenre-extracted with dichloromethane (2×200 ml). Organic extracts werecombined, dried over anhydrous magnesium sulphate, filtered andconcentrated in vacuo to give the title compound as a colourless oilthat crystallised on standing to give colourless crystals (15.3 g, 0.07mol, 59%). ¹H NMR (CDCl₃, 400 MHz) δ: 0.93 (t, 3H), 1.62 (q, 2H), 2.71(q, 2H), 2.81 (t, 2H), 3.00 (d, 1H), 3.80 (s, 3H), 4.30 (bs, 1H), 4.89(d, 1H), 6.81 (d, 1H), 6.91 (d, 1H), 6.93 (s, 1H), 7.22 (t, 1H). LRMS:m/z 210. Mpt: 50-51° C. Analysis found C, 67.47; H, 9.02; N, 6.45%.C₁₂H₁₉NO₂.0.2H₂O requires C, 67.70; H, 9.19; N, 6.58%.

EXAMPLE 42-Chloro-N-[2-hydroxy-2-(3-methoxyphenyl)ethyl]-N-propylacetamide

Sodium hydroxide (15.1 g, 0.38 mol) in water (180 ml) was added to theamine from example 3 (15.8 g, 0.08 mol) in dichloromethane (500 ml) andthe solution vigorously stirred at room temperature.Chloroacetylchloride (7.22 ml, 0.09 mol) was then added and the reactionmixture stirred for a further 30 minutes. The layers were separated andthe aqueous layer re-extracted with dichloromethane (200 ml). Theorganic extracts were combined, dried over anhydrous magnesium sulphate,filtered and concentrated in vacuo to give the title compound as acolourless oil (17.8 g, 0.06 mol, 83%). ¹H NMR (CDCl₃, 400 MHz) δ: 0.96(t, 3H), 1.62 (q, 2H), 3.21 (q, 2H), 3.57-3.71 (m, 2H), 3.82 (s, 3H),4.01-4.21 (bq, 1H), 4.16 (s, 2H), 5.00 (m, 1H), 6.82 (m, 1H), 6.91-6.99(m, 2H), 7.22 (m, 1H). LRMS: m/z 286. Analysis found C, 57.38; H, 6.95;N, 4.67%. C₁₄H₂₀NO₃Cl.0.33H₂O requires C, 57.64; H, 7.14; N, 4.80%.

EXAMPLE 5 6-(3-Methoxyphenyl)-4-Propylmorpholin-3-one

Potassium hydroxide (4.2 g, 0.07 mol), isopropyl alcohol (500 ml) andthe amide from example 4 (17.8 g, 0.06 mol) were stirred together as anopaque solution with water (15 ml) for 2 hours. The reaction mixture wasconcentrated in vacuo and the yellow residue dissolved in ethyl acetate(200 ml). This was partitioned with water (200 ml) then brine (200 ml).The organic fraction was dried over anhydrous magnesium sulphate,filtered and concentrated in vacuo to give the title compound as ayellow oil (15.8 g, 0.06 mol, 100%). ¹H NMR (CDCl₃, 400 MHz) δ: 0.96 (t,3H), 1.62 (m, 2H), 3.36 (m, 2H), 3.51 (q, 2H), 3.81 (s, 3H), 4.30-4.62(bq, 2H), 4.79 (d, 1H), 6.85 (d, 1H), 6.91 (d, 1H), 6.95 (s, 1H), 7.29(t, 1H). LRMS: m/z 272. Analysis found C, 66.80; H, 7.78; N, 5.52%.C₁₄H₁₉NO₃.0.1H₂O requires C, 66.96; H, 7.71; N, 5.58%.

EXAMPLE 6 2-(3-Methoxyphenyl)-4-propylmorpholine

Borane-tetrahydrofuran complex (1M in THF) (200 ml, 0.19 mol) was addeddropwise to the morpholin-3-one from example 5 (15.8 g, 0.06 mol) in dryTHF (100 ml) under an atmosphere of nitrogen, over 30 minutes. Thereaction mixture was brought to reflux for 3 hours then cooled andquenched by addition of methanol (30 ml). The reaction mixture was thenconcentrated in vacuo and the colourless residue cautiously suspended in4N HCl (aq) (400 ml) before refluxing for 2.5 hours. The reactionmixture was cooled and dichloromethane (200 ml) added. Layers wereseparated and the aqueous layer rendered basic by addition of potassiumcarbonate before re-extracting with dichloromethane (3×100 ml). Theorganic extracts were combined, dried over anhydrous magnesium sulphate,filtered and concentrated in vacuo to give the title compound as acolourless oil (12.51 g, 0.05 mol, 84%). ¹H NMR (CDCl₃, 400 MHz) δ: 0.95(t, 3H), 1.59 (q, 2H), 2.05 (t, 1H), 2.23 (t, 1H), 2.40 (t, 2H), 2.81(d, 1H), 2.98 (d, 1H), 3.80 (s, 3H), 3.85 (t, 1H), 4.05 (d, 1H), 4.60(d, 1H), 6.81 (d, 1H), 6.91 (d, 1H), 7.21 (t, 1H), 7.23 (s, 1H). LRMS:n/z 236. Analysis found C, 68.94; H, 8.80; N, 5.79%. C₁₄H₂₁NO₂.0.5H₂Orequires C, 68.82; H, 9.08; N, 5.73%.

EXAMPLE 7A R-(−)-3-(4-Propylmorpholin-2-yl)phenol EXAMPLE 7BS-(+)-3-(4-Propylmorpholin-2-yl)phenol

Hydrobromic acid (250 ml) and the anisole from example 6 (8.62 g, 0.03mol) were heated to reflux together for 1 hour. After cooling thereaction mixture was diluted with water (100 ml) then neutralised byaddition of NH₄OH (20 ml). The yellow opaque solution was then extractedwith dichloromethane (2×100 ml). The organic extracts were combined thendried over anhydrous magnesium sulphate, filtered and concentrated invacuo to give the racemic mixture of the title compound as a yellow oil(7.78 g, 0.03 mol, 96%). The enantiomers were separated by chiralchromatography (Chiralpak AD 250*20 mm column) eluting withhexane:isopropyl alcohol:diethylamine (70:30:0.05) to give enantiomer 1(ee>99.5%) and enantiomer 2 (ee>99%). Each enantiomer was purified bycolumn chromatography on silica eluting with dichloromethane:methanol(95:5) to give enantiomer 1(7a) (3.02 g, 0.014 mol, 39%) and enantiomer2 (7b) (3.15 g, 0.014 mol, 40%) as colourless oils. Enantiomer 1 (7a):¹H NMR (CDCl₃, 400 MHz) δ: 0.96 (t, 3H), 1.60 (q, 2H), 2.13 (t, 1H),2.31 (t, 1H), 2.41 (t, 2H), 2.85 (d, 1H), 3.02 (d, 1H), 3.90 (t, 1H),4.02 (dd, 1H), 4.60 (d, 1H), 6.78 (d, 1H), 6.80 (s, 1H), 6.91 (d, 1H),7.20 (t, 1H). LRMS: m/z 222 (M−H⁺). Enantiomer 2 (7b): ¹H NMR (CDCl₃,400 MHz) δ: 0.96 (t, 3H), 1.60 (q, 2H), 2.13 (t, 1H), 2.31 (t, 1H), 2.41(t, 2H), 2.85 (d, 1H), 3.02 (d, 1H), 3.90 (t, 1H), 4.02 (dd, 1H), 4.60(d, 1H), 6.78 (d, 1H), 6.80 (s, 1H), 6.91 (d, 1H), 7.20 (t, 1H). LRMS:m/z 222 (M−H⁺).

EXAMPLE 8 R-(−)-3-(4-Propylmorpholin-2-yl)phenol hydrochloride

Enantiomer 1 (7a) of example 7 (3.00 g, 0.014 mol) was dissolved indiethyl ether (180 ml) and hydrogen chloride (2.0M solution in diethylether) (10 ml) was added. The reaction mixture was stirred at roomtemperature for 30 minutes, then the solvent was decanted and dried invacuo, giving title compound as a white solid (3.115 g, 0.012 mol, 90%).¹H NMR (CD₃OD, 400 MHz) δ: 1.06 (t, 3H), 1.81 (m, 2H), 3.02 (t, 1H),3.16 (t, 2H), 3.20 (t, 1H), 3.60 (t, 2H), 4.01 (t, 1H), 4.26 (d, 1H),4.71 (d, 1H), 6.78 (d, 1H), 6.82 (s, 1H), 6.83 (d, 1H), 7.21 (t, 1H).LRMS: m/z 222 (M−H⁺). Analysis found C, 59.74; H, 7.98; N, 5.25%.C₁₃H₁₉NO₂.0.18H₂O requires C, 59.82; H, 7.86; N, 5.37%. α_(D)=−5.66°(Methanol 10.6 mg/10 ml).

A SAMPLE OF THE TITLE COMPOUND WAS RE CRYSTALLISED BY VAPOUR DIFFUSIONUSING A METHANOL: DIETHYL ETHER MIX AND AN X-RAY CRYSTAL STRUCTUREOBTAINED. THE ABSOLUTE STEREOCHEMISTRY OF THE TITLE COMPOUND WASDETERMINED FROM THE DIFFRACTION DATA BY THE METHOD OF FLACK¹ AND WASSHOWN TO HAVE AN ‘R’ CONFIGURATION. REF 1: H. D. FLACK, ACTA CRYST.1983, 439, 876-881

EXAMPLE 9 2-Amino-1-(3,5-dimethoxyphenyl)ethanol

Prepared following the same method as for example 1 starting from3,5-dimethoxybenzaldehyde (5.00 g, 0.03 mol). After refluxing in 6M HCl(aq) the reaction mixture was cooled and extracted with diethyl ether(2×80 ml). The organic layers were discarded and the aqueous layerbasified by the addition of potassium carbonate. The aqueous residue wasthen extracted with ethyl acetate (3×70 ml). The organic extracts werecombined and dried over anhydrous magnesium sulphate, filtered andconcentrated in vacuo to give the title compound as a pale yellow oil(3.47 g, 0.018 mol, 59%). ¹H NMR (CD₃OD, 400 MHz) δ: 2.77-2.86 (m, 2H),3.78 (s, 6H), 4.60 (m, 1H), 6.38 (s, 1H), 6.52 (s, 2H). LRMS: m/z 198(M−H⁺).

EXAMPLE 10 N-[2-(3,5-dimethoxyphenyl)-2-hydroxyethyl]propionamide

Prepared following the same method as for example 2 starting from theamine in example 9 (3.41 g, 0.017 mol). The crude reaction mixture waspurified by column chromatography on silica eluting withdichloromethane:methanol (95:5) to give the title compound as a brightyellow oil (3.08 g, 0.012 mol, 70%). ¹H NMR (CDCl₃, 400 MHz) δ: 1.18 (m,3H), 2.24 (m, 2H), 3.34 (m, 1H), 3.68 (m, 1H), 3.81 (s, 6H), 4.80 (dd,1H), 5.95 (bs, 1H), 6.39 (s, 1H), 6.51 (s, 2H). LRMS: m/z 252 (M−H⁻).

EXAMPLE 11 1-(3,5-dimethoxyphenyl)-2-propylaminoethanol

Prepared following the method as for example 3 starting from the amidein example 10 (3.06 g, 0.012 mol) to give the title compound as anorange oil (2.72 g, 0.011 mol, 94%). ¹H NMR (CD₃OD, 400 MHz) δ: 0.95 (t,3H), 1.56 (m, 2H), 2.61 (m, 2H), 2.77 (d, 2H), 3.78 (s, 6H), 4.70 (t,1H), 6.38 (s, 1H), 6.51 (s, 2H). LRMS: m/z 240 (M−H⁺).

EXAMPLE 122-Chloro-N-[2-(3,5-dimethoxyphenyl)-2-hydroxyethyl]-N-propylacetamide

Prepared following the same method as for example 4 starting from theamine in example 11 (2.70 g, 0.011 mol) to give the title compound as ayellow oil (3.56 g, 0.011 mol, 100%). ¹H NMR (CDCl₃, 400 MHz) δ: 0.92(t, 3H), 1.61 (m, 2H), 3.20 (m, 2H), 3.51-3.64 (m, 2H), 3.80 (d, 6H),4.13 (s, 2H), 4.95 (m, 1H), 6.40 (m, 1H), 6.55 (s, 2H). LRMS: m/z 316(M−H⁺).

EXAMPLE 13 6-(3,5-Dimethoxyphenyl)-4-propylmorpholin-3-one

Prepared following the same method as for example 5 starting from theamide in example 12 (3.54 g, 0.011 mol) to give the title compound as ayellow oil (2.44 g, 0.009 mol, 78%). ¹H NMR (CDCl₃, 400 MHz) δ: 0.94 (t,3H), 1.61 (m, 2H), 3.30 (m, 2H), 3.49 (m, 2H), 3.80 (s, 6H), 4.30 (d,1H), 4.42 (d, 1H), 4.73 (dd, 1H), 6.42 (s, 1H), 6.53 (s, 2H). LRMS: m/z280 (M−H⁺).

EXAMPLE 14 2-(3,5-Dimethoxyphenyl)-4-propylmorpholine

Prepared following the method as for example 6 starting from the amidein example 13 (2.42 g, 0.009 mol). After refluxing in 6M HCl (aq) thecooled reaction mixture was extracted with diethyl ether (2×80 ml). Theorganic layers were discarded and the aqueous basified by addition ofpotassium carbonate. The aqueous residue was then extracted with ethylacetate (3×80 ml) and the organic extracts combined, dried overanhydrous magnesium. sulphate, filtered then concentrated in vacuo togive the title compound as a pale orange oil (2.14 g, 0.008 mol, 93%).¹H NMR (CD₃OD, 400 MHz) δ: 0.95 (t, 3H), 1.58 (m, 2H), 2.01 (m, 1H),2.22 (dt, 1H), 2.38 (t, 2H), 2.83 (d, 1H), 2.93 (d, 1H), 3.78 (m, 7H),4.01 (dd, 1H), 4.45 (dd, 1H), 6.39 (s, 1H), 6.49 (s, 2H). LRMS: m/z 266(M−H⁺).

EXAMPLE 15A R-5-(4-Propylmorpholin-2-yl)benzene-1,3-diol EXAMPLE 15BS-5-(4-Propylmorpholin-2-yl)benzene-1,3-diol

Prepared following the same route as for example 7 starting from the3,5-dimethoxyphenyl compound in example 14 (1.00 g, 0.004 mol) givingthe title racemic compound as a brown oil (145 mg, 0.61 mmol, 16%). Theenantiomers were separated by chiral chromatography (Chiralpak AD 250*20 mm column) eluting with hexane:isopropyl alcohol: (80:20) to giveenantiomer 1 (15a) (5.2 mg) (ee>98.94%) and enantiomer 2 (15b) (5.1 mg)(ee>96.46%) as brown oils. Enantiomer 1 (15a): ¹H NMR (CD₃OD, 400 MHz)δ: 0.96 (t, 3H), 1.58 (m, 2H), 2.01 (t, 1H), 2.20 (dt, 1H), 2.37 (t,2H), 2.81-2.92 (m, 2H), 3.89 (dt, 1H), 3.99 (dd, 1H), 4.38 (dd, 1H),6.18 (t, 1H), 6.26 (s, 2H). LRMS: m/z 238 (M−H⁺). Enantiomer 2 (15b): ¹HNMR (CD₃OD, 400 MHz) δ: 0.95 (t, 3H), 1.58 (m, 2H), 2.01 (t, 1H), 2.20(dt, 1H), 2.38 (t, 2H), 2.80-2.92 (q, 2H), 3.78 (dt, 1H), 3.98 (dd, 1H),4.38 (dd, 1H), 6.18 (s, 1H), 6.25 (s, 2H). LRMS: m/z 238 (M−H⁺).

EXAMPLE 16 4-Fluoro-3-methoxybenzaldehyde

(4-Fluoro-3-methoxyphenyl) methanol (5.00 g, 0.03 mol) and manganesedioxide (33.4 g, 0.38 mol) were stirred in dichloromethane (100 ml)under an atmosphere of nitrogen, at gentle reflux for 16 hours. Thecooled reaction mixture was then filtered through arbacel andconcentrated in vacuo to give the title compound as a white solid (4.18g, 0.027 mol, 85%). ¹H NMR (CDCl₃, 400 MHz) δ: 3.96 (s, 3H), 7.23 (d,1H), 7.43 (m, 1H), 7.50 (d, 1H) 9.91 (s, 1H). Mpt: 61-63° C. Analysisfound C, 62.18; H, 4.54%. C₈H₇FO₂ requires C, 62.34; H, 4.58%.

EXAMPLE 17 2-Amino-1-(4-fluoro-3-methoxyphenyl)ethanol

Prepared following the same method as for example 1 starting from4-fluoro-3-methoxybenzaldehyde (4.17 g, 0.03 mol). After refluxing in 6MHCl (aq) the reaction mixture was cooled and extracted with diethylether (2×60 ml). The organic layers were discarded and the aqueous layerbasified by the addition of potassium carbonate. The aqueous residue wasthen extracted with ethyl acetate (3×80 ml). The organic extracts werecombined and dried over anhydrous magnesium sulphate, filtered andconcentrated in vacuo to give the title compound as an orange oil (2.36g, 0.013 mol, 47%). ¹H NMR (CD₃OD, 400 MHz) δ: 2.80-2.91 (m, 2H), 3.86(s, 3H), 4.64 (m, 1H), 6.89 (m, 1H), 7.03 (t, 1H), 7.11 (dd, 1H). LRMS:m/z 186 (M−H⁺).

EXAMPLE 18 N-[2-(4-Fluoro-3-methoxyphenyl)-2-hydroxyethyl]propionamide

Prepared following the same method as for example 2 starting with theamine from example 17 (1.32 g, 0.007 mol). The crude reaction mixturewas purified by column chromatography on silica eluting with ethylacetate:pentane (2:1) to give the title compound as a yellow oil thatcrystallised on standing (0.59 g, 0.002 mol, 35%). ¹H NMR (CDCl₃, 400MHz) δ: 1.18 (t, 3H), 2.24 (q, 2H), 2.58 (bs, 1H), 3.34 (m, 1H), 3.63(m, 1H), 3.88 (s, 3H), 4.82 (dd, 1H), 5.98 (bs, 1H), 6.82 (m, 1H), 7.01(m, 2H). LRMS: m/z 242 (M−H⁺).

EXAMPLE 19 1-(4-Fluoro-3-methoxyphenyl)-2-propylaminoethanol

Prepared following the same method as for example 3 starting with theamide from example 18 (585 mg, 2.42 mmol). After refluxing in 6M HCl(aq) the reaction mixture was cooled and extracted with diethyl ether(2×50 ml). The organic layers were discarded and the aqueous layerbasified by the addition of potassium carbonate. The aqueous residue wasthen extracted with ethyl acetate (3×50 ml). The organic extracts werecombined and dried over anhydrous magnesium sulphate, filtered andconcentrated in vacuo to give the title compound as a pale yellow oil(448 mg, 1.97 mmol, 81%). ¹H NMR (CD₃OD, 400 MHz) δ: 0.96 (t, 3H), 1.58(m, 2H), 2.63 (m, 2H), 2.79 (d, 2H), 3.96 (s, 3H), 4.77 (t, 1H), 6.90(m, 1H), 7.03 (t, 1H), 7.11 (d, 1H). LRMS: m/z 228 (M−H⁺).

EXAMPLE 202-Chloro-N-[2-(4-fluoro-3-methoxyphenyl)-2-hydroxyethyl]-N-propylacetamide

Prepared following the same method as for example 4 starting with theamine from example 19 (0.84 g, 4.00 mmol) to give the title compound asa yellow oil (0.97 g, 3.00 mmol, 87%). LRMS: m/z 304 (M−H⁺). This wastaken on crude.

EXAMPLE 21 6-(4-Fluoro-3-methoxyphenyl)-4-propylmorpholin-3-one

Prepared following the same method as for example 5 starting with theamide from example 20 (0.96 g, 3.00 mmol) to give the title compound asa yellow oil (0.64 g, 2.40 mmol, 75%). ¹H NMR (CDCl₃, 400 MHz) δ: 0.94(t, 3H), 1.62 (m, 2H), 3.33 (m, 2H), 3.48 (m, 2H), 3.91 (s, 3H), 4.34(d, 1H), 4.43 (d, 1H), 4.76 (dd, 1H), 6.85 (m, 1H), 7.01-7.08 (m, 2H).LRMS: m/z 268 (M−H⁺).

EXAMPLE 22 2-(4-Fluoro-3-methoxyphenyl)-4-propylmorpholine

Prepared following the same method as for example 6 starting with themorpholin-3-one from example 21 (633 mg, 2.37 mmol). After refluxing in6M HCl (aq) the reaction mixture was cooled and extracted with diethylether (2×20 ml). The organic layers were discarded and the aqueous layerbasified by the addition of potassium carbonate. The aqueous residue wasthen extracted with ethyl acetate (3×20 ml). The organic extracts werecombined and dried over anhydrous magnesium sulphate, filtered andconcentrated in vacuo to give the title compound as a yellow oil (552mg, 2.18 mmol, 92%). ¹H NMR (CD₃OD, 400 MHz) δ: 0.95 (t, 3H), 1.58 (m,2H), 2.02 (t, 1H), 2.22 (dt, 1H), 2.38 (t, 2H), 2.85 (d, 1H), 2.93 (d,1H), 3.80 (m, 1H), 3.84 (s, 3H), 4.01 (dd, 1H), 4.50 (dd, 1H), 6.88 (m,1H), 7.02 (t, 1H), 7.09 (d, 1H). LRMS: m/z 254 (M−H⁺).

EXAMPLE23A R-(+)-2-Fluoro-5-(4-propylmorpholin-2-yl)phenol EXAMPLE 23BS-(−)-2-Fluoro-5-(4-propylmorpholin-2-yl)phenol

Prepared following the same method as for example 7 starting with theanisole from example 22 (200 mg, 0.789 mmol). The crude reaction mixturewas purified by column chromatography on silica eluting withdichloromethane: methanol (90:10) to give the title racemic compound asa dark yellow viscous oil (149 mg, 0.62 mmol, 79%). The enantiomers wereseparated by chiral chromatography (Chiralpak AD 250*20 mm column)eluting with hexane:isopropyl alcohol: (90:10) to give enantiomer 1(23a) as an opaque oil (15 mg) (ee>99.5%) and enantiomer 2 (23b) as acrystalline solid (16 mg) (ee>99%). Enantiomer 1 (23a): ¹H NMR (CD₃OD,400 MHz) δ: 0.95 (t, 3H), 1.58 (m, 2H), 2.01 (t, 1H), 2.21 (dt, 1H),2.37 (t, 2H), 2.82-2.97 (bq, 2H), 3.78 (dt, 1H), 3.99 (dd, 1H), 4.43 (d,1H), 6.78 (m, 1H), 6.89-7.01 (m, 2H). LRMS: m/z 240 (M−H⁺). α_(D)=+0.91(Ethanol 1.10 mg/ml). Enantiomer 2 (23b): ¹H NMR (CD₃OD, 400 MHz) δ:0.96 (t, 3H), 1.58 (m, 2H), 2.01 (t, 1H), 2.22 (dt, 1H), 2.38 (t, 2H),2.78 (dd, 2H), 3.78 (dt, 1H), 4.00 (dd, 1H), 4.43 (dd, 1H), 6.78 (m,1H), 6.91 (d, 1H), 6.98 (t, 1H). LRMS: m/z 240 (M−H⁺). α_(D)=−0.40(Ethanol 1.00 mg/ml).

EXAMPLE 24 2-Amino-1-(4-benzyloxyphenyl)ethanol

Potassium cyanide (20.15 g, 0.31 mol) and ammonium chloride (16.4 g,0.31 mol) were dissolved in water (60 ml) to which was added4-benzyloxybenzaldehyde (32.9 g, 0.155 mol) followed by diethyl ether(100 ml). The reaction mixture was stirred vigorously for 48 hours atroom temperature before extracting with ethyl acetate (2×200 ml). Thecombined organic layers were dried over anhydrous magnesium sulphate,filtered and concentrated in vacuo to give the cyanohydrin intermediateas a yellow solid (34.2 g, 0.14 mol, 90%). The cyanohydrin was thendissolved in dry THF (300 ml) and borane-methyl sulphide complex (26.6ml, 0.28 mol) was added. The reaction mixture was refluxed for 2 hoursbefore being quenched with methanol (50 ml). Water (50 ml) was addedfollowed by c.HCl (40 ml) and the reaction mixture was stirred for 2hours until the exotherm subsided. The reaction mixture was thenconcentrated in vacuo and the residue diluted with water (100 ml). Theaqueous solution was then basified by addition of NH₄OH (30 ml), andextracted with ethyl acetate (3×150 ml). The organic extracts were driedover anhydrous magnesium sulphate, filtered and concentrated in vacuo togive the title compound as a white solid (24.8 g, 0.10 mol, 73%). ¹H NMR(CDCl₃, 400 MHz) δ: 1.62 (bs, 3H), 2.81 (dd, 1H), 2.99 (d, 1H), 4.61 (q,1H), 5.07 (s, 2H), 6.95 (d, 2H), 7.22-7.45 (m, 7H). LRMS: m/z 244(M−H⁺).

EXAMPLE 25 N-[2-(4-benzyloxylphenyl)-2-hydroxyethyl)propionamide

The amine from example 24 (24.8 g, 0.10 mol) was dissolved indichloromethane (700 ml) and to this was added triethylamine (20.86 ml,0.15 mol). The reaction mixture was stirred and cooled to 0° C., beforepropionyl chloride (7.12 ml, 0.082 mol) was added dropwise. The reactionmixture was then allowed to warm to room temperature over 16 hoursbefore quenching with 3M HCl (aq) (20 ml) and water (100 ml). Thereaction mixture was then extracted with dichloromethane (3×200 ml) andthe combined organic layers dried over anhydrous magnesium sulphate,filtered and concentrated in vacuo to give the title compound as a clearviscous gum (27.5 g, 0.092 mol, 90%). ¹H NMR (CDCl₃, 400 MHz) δ: 1.10(t, 3H), 2.19 (q, 2H), 3.32-3.43 (m, 4H), 4.81 (s, 2H), 5.11 (m, 1H),6.99 (d, 2H), 7.25-7.42 (m, 7H). LRMS: m/z 298 (M−H⁻).

EXAMPLE 26 1-(4-benzyloxyphenyl)-2-propylaminoethanol

To the amide from example 25 (27.5 g, 0.092 mol) in dry THF (100 ml) wasadded borane-methyl sulphide complex (17.5 ml, 0.18 mol) and thereaction mixture was stirred at reflux for 2 hours. The reaction mixturewas cooled then quenched with methanol (30 ml). Water (50 ml) and c.HCl(35 ml) were added and the reaction mixture stirred until all bubblingceased before concentrating in vacuo. To the residue water (250 ml) wasadded, before basifying by addition of NH₄OH (30 ml). The aqueous layerwas extracted with ethyl acetate (3×200 ml) and the combined organicextracts dried over anhydrous magnesium sulphate, filtered andconcentrated in vacuo to give the title compound as a white solid (26.1g, 0.09 mol, 99%). ¹H NMR (CD₃OD, 400 MHz) δ: 0.95 (t, 3H), 1.58 (q,2H), 2.62 (m, 2H), 2.81 (m, 2H), 4.72 (dd, 1H), 5.05 (s, 2H), 6.95 (d,2H), 7.24 (m, 3H), 7.35 (t, 2H), 7.41 (d, 2H). LRMS: m/z 286 (M−H⁺).

EXAMPLE 27 6-(4-benzyloxyphenyl)-4-propylmorpholin-3-one

Sodium hydroxide (22.5 g, 0.56 mol) in water (100 ml) was added to theamine from example 26 (26.0 g, 0.09 mol) in dichloromethane (400 ml) andthe solution vigorously stirred at room temperature.Chloroacetylchloride (8.6 ml, 0.11 mol) was then added and the reactionmixture stirred for a further 60 minutes. The layers were separated andthe aqueous layer re-extracted with dichloromethane (200 ml). Theorganic extracts were combined, dried over anhydrous magnesium sulphate,filtered and concentrated in vacuo to give a colourless oil. Potassiumhydroxide (15.0 g, 0.27 mol), isopropyl alcohol (400 ml) and thecolourless oil residue were stirred together as an opaque solution withwater (30 ml) for 2 hours. The reaction mixture was concentrated invacuo and the yellow residue dissolved in ethyl acetate (200 ml). Thiswas partitioned with water (200ml) then brine (200 ml). The organicfraction was dried over anhydrous magnesium sulphate, filtered andconcentrated in vacuo to give the title compound as a white solid (19.9g, 0.06 mol, 67%). ¹H NMR (CDCl₃, 400 MHz) δ: 0.95 (t, 3H), 1.62 (m,2H), 3.34 (m, 2H), 3.51 (m, 2H), 4.32 (d, 1H), 4.41 (d, 1H), 4.72 (dd,1H), 5.04 (s, 2H), 6.98 (d, 2H), 7.31-7.43 (m, 7H). LRMS: m/z 326(M−H⁺).

EXAMPLE 28 2-(4-benzyloxyphenyl)-4-propylmorpholine

Prepared following the same method as for example 26 with themorpholin-3-one from example 27 (19.9 g, 0.061 mol) to give the titlecompound as a colourless oil (17 g, 0.055 mol, 90%). ¹H NMR (CDCl₃, 400MHz) δ: 0.95 (t, 3H), 1.55 (q, 2H), 2.06 (t, 1H), 2.21 (dt, 1H), 2.35(dd, 2H), 2.80 (d, 1H), 2.91 (d, 1H), 3.82 (dt, 1H), 4.02 (dd, 1H), 4.52(dd, 1H), 5.05 (s, 2H), 6.98 (t, 2H), 7.24-7.42 (m, 7H). LRMS: m/z 312(M−H⁺).

EXAMPLE 29 4-(4-Propylmorpholin-2-yl)phenol

Benzyl ether from example 28 (3.0 g, 9.64 mmol) was dissolved inmethanol (150 ml) and 10% palladium on charcoal (800 mg) was added. Thereaction mixture was stirred for a few minutes before ammonium formate(6.17 g, 96.4 mmol) was added portionwise. The reaction mixture wascarefully heated to 80° C. until gas evolution had ceased. Aftercooling, the reaction mixture was filtered through arbacel, washed withmethanol (50 ml) and concentrated in vacuo to give the title compound asa white crystalline solid (1.51 g, 6.83 mmol, 71%). ¹H NMR (CDCl₃, 400MHz) δ: 0.91 (t, 3H), 1.58 (q, 2H), 2.10 (t, 1H), 2.22 (t, 1H), 2.40(dd, 2H), 2.81 (d, 1H), 2.93 (d, 1H), 3.85 (t, 1H), 4.02 (dd, 1H), 4.57(d, 1H), 6.79 (d, 2H), 7.21 (d, 2H). LRMS: m/z 222 (M−H⁺).

EXAMPLE 30 2-Bromo-4-(4-propylmorpholin-2-yl)phenol

To the phenol from example 29 (200 mg, 0.9 mmol) in dichloromethane (5ml) was added N-bromosuccinimide (161 mg, 0.9 mmol). The reactionmixture was stirred at room temperature for 55 hours, beforeconcentrating in vacuo. The crude product was purified by columnchromatography on silica eluting with dichloromethane:methanol (95:5) togive the title compound as a white foam (117.5 mg, 0.39 mmol, 44%). ¹HNMR (CDCl₃, 400 MHz) δ: 0.96 (t, 3H), 1.59 (q, 2H), 2.03 (t, 1H), 2.23(t, 1H), 2.40 (t, 2H), 2.81 (d, 1H), 2.98 (d, 1H), 3.82 (t, 1H), 4.01(d, 1H), 4.56 (d, 1H), 6.96 (d, 1H), 7.20 (d, 1H), 7.49 (s, 1H). LRMS:m/z 302 (M−H⁺, Br isotope).

EXAMPLE 31 2-(4-benzyloxy-3-bromophenyl)-4-propylmorpholine

To the phenol from example 30 (117.5 mg, 0.39 mmol) in dry DMF (10 ml),under an atmosphere of nitrogen, was added potassium carbonate (75 mg,0.54 mmol) and benzyl bromide (0.07 ml, 0.54 mmol). The reaction mixturewas heated to 150° C. for 48 hours. After cooling, the reaction mixturewas concentrated in vacuo and the residue partitioned between ethylacetate (50 ml) and water (50 ml). The aqueous layer was thenre-extracted with ethyl acetate (2×20 ml). The combined organic extractswere then dried over anhydrous magnesium sulphate, filtered andconcentrated in vacuo to give the crude product as a brown oil. This waspurified by column chromatography on silica eluting withdichloromethane:methanol (98:2) to give the title compound as acolourless oil (153 mg, 0.39 mmol, 100%). ¹H NMR (CDCl₃, 400 MHz) δ:0.93 (t, 3H), 1.56 (q, 2H), 2.05 (t, 1H), 2.25 (t, 1H), 2.37 (t, 2H),2.82 (d, 1H), 2.92 (d, 1H), 3.85 (t, 1H), 4.02 (d, 1H), 4.52 (d, 1H),5.15 (s, 2H), 6.87 (d, 1H), 7.20 (d, 1H), 7.30 (d, 1H), 7.37 (t, 2H),7.45 (d, 2H), 7.58 (s, 1H). LRMS: m/z 392 (M−H⁺).

EXAMPLE 32 2-Benzyloxy-5-(4-propylmorpholin-2-yl)benzoic acid methylester

To the bromide from example 31 (153 mg, 0.39 mmol) in dry DMF (4 ml) wasadded triethylamine (2.1 ml, 0.78 mmol) and methanol (2 ml) and thereaction mixture stirred for 5 minutes.[1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium (II), complexwith dichloromethane (1:1) (16 mg, 0.02 mmol) was added before carbonmonoxide (g) (3 inflated balloons) was bubbled through the reactionmixture. The reaction mixture was then heated to 100° C. for 16 hoursunder an atmosphere of carbon monoxide. After cooling, the reactionmixture was concentrated in vacuo and the residue partitioned betweenethyl acetate (25 ml) and water (20 ml). The organic layer wasseparated, washed with brine (20 ml) and dried over anhydrous magnesiumsulphate, filtered and concentrated in vacuo to give a black solid.Purification by column chromatography on silica eluting withdichloromethane:methanol:ammonia (90:10:1) gave the title compound as acolourless oil (105 mg, 0.28 mmol, 73%). ¹H NMR (CDCl₃, 400 MHz) δ: 0.94(t, 3H), 1.60 (m, 2H), 2.18 (s, 4H), 2.43 (m, 2H), 3.00 (m, 2H), 3.90(s, 3H), 4.04 d, 1H), 5.18 (s, 2H), 5.97 (d, 1H), 7.26-7.47 (m, 6H),7.82 (s, 1H). LRMS: m/z 370 (M−H⁺).

EXAMPLE 33 2-Benzyloxy-5-(4-propylmorpholin-2-yl)benzoic acid

To the methyl ester from example 32 (105 mg, 0.28 mmol) in methanol (5ml) was added 10% sodium hydroxide (aq) (15 ml) and the milky whitesuspension was refluxed for 2 hours. The now colourless reaction mixturewas cooled then neutralised by addition of 2M HCl (aq) (few drops). Thereaction mixture was then concentrated in vacuo to give the titlecompound as an off-white solid (99 mg, 0.28 mmol, 100%). LRMS: m/z 355(M−H⁺). This material was taken on crude to example 34.

EXAMPLE 34 2-Benzyloxy-5-(4-propylmorpholin-2-yl)benzamide

To the crude benzoic acid from example 33 (99 mg, 0.28 mmol) was addedthionyl chloride (5 ml) and the reaction mixture heated to 50° C. for 2hours. The reaction mixture was cooled and the excess thionyl chloridewas removed in vacuo. The residue was then dissolved in dichloromethane(10 ml) and ammonia (g) was bubbled through the reaction mixture for 10minutes. The resulting suspension was stirred at room temperature for 1hour before concentrating in vacuo. The crude material was purified bycolumn chromatography on silica eluting withdichloromethane:methanol:ammonia (95:5:0.5) to give the title compoundas an off-white solid (88 mg, 0.25 mmol, 90%). ¹H NMR (CDCl₃, 400 MHz)δ: 0.94 (t, 3H), 1.59 (m, 2H), 2.15-2.42 (m, 4H), 2.87 (m, 1H), 3.03 (m,1H), 3.96 (m, 1H), 4.02 (d, 1H), 4.67 (m, 1H), 5.19 (s, 2H), 5.72 (m,1H), 7.04 (d, 1H), 7.41 (m, 5H), 7.50 (d, 1H), 7.70 (m, 1H), 8.21 (s,1H). LRMS: m/z 355 (M−H⁺).

EXAMPLE 35 2-Hydroxy-5-(4-propylmorpholin-2-yl)benzamide

Prepared using the same method as for example 29 with the benzyl esterfrom example 34 (80 mg, 0.22 mmol) to give the title compound as anoff-white solid (56 mg, 0.21 mmol, 96%). ¹H NMR (CD₃OD, 400 MHz) δ: 0.95(t, 3H), 1.55 (m, 2H), 2.13 (t, 1H), 2.29 (t, 1H), 2.42 (m, 2H), 2.88(d, 1H), 2.97 (d, 1H), 3.81 (t, 1H), 4.00 (d, 1H), 4.49 (d, 1H), 6.87(d, 1H), 7.42 (d, 1H), 7.78 (s, 1H). LRMS: m/z 265 (M−H⁺).

EXAMPLE 36 2-Nitro-4-(4-propylmorpholin-2-yl)phenol

The phenol from example 29 (100 mg, 0.45 mmol) was dissolved in nitricacid:water (1:3) (2 ml) and stirred at room temperature for 10 minutes.The reaction mixture was then diluted with water (5 ml) and basifiedwith NH₄OH (1 ml), before extracting into ethyl acetate (3×10 ml). Theorganic extracts were combined and dried over anhydrous magnesiumsulphate, filtered and concentrated in vacuo to give the title compoundas a yellow solid (95 mg, 0.35 mmol, 79%). ¹H NMR (CDCl₃, 400 MHz) δ:0.97 (t, 3H), 1.33 (t, 2H), 1.43-1.79 (bm, 4H), 2.02 (d, 3H), 4.06 (m,2H), 7.17 (d, 1H), 7.60 (d, 1H), 8.16 (s, 1H), 10.55 (bs, 1H). LRMS: m/z267 (M−H⁺).

EXAMPLE 37 2-Amino-4-(4-propylmorpholin-2-yl)phenol

To the nitro from example 36 (95 mg, 0.35 mmol) in ethanol (10 ml) wasadded 10% palladium on charcoal (50 mg) and ammonium formate (100 mg,XS). The reaction mixture was gently heated to 70° C. and held at thistemperature for 1 hour before it was allowed to cool to roomtemperature. The reaction mixture was then filtered through arbacel andwashed with ethanol (20 ml) then dichloromethane (20 ml). The organicwashes were combined and concentrated in vacuo to give the titlecompound as a yellow solid (65 mg, 0.28 mmol, 78%). ¹H NMR (CDCl₃, 400MHz) δ: 0.91 (t, 3H), 1.55 (m, 2H), 2.12 (t, 1H), 2.25 (dt, 1H), 2.40(t, 2H), 2.81-2.92 (dd, 2H), 3.82 (t, 1H), 4.00 (d, 1H), 4.42 (d, 1H),6.60 (m, 2H), 6.71 (s, 1H). LRMS: m/z 237 (M−H⁺).

EXAMPLE 38 5-Bromo-2-(2,5-dimethylpyrrol-1-yl pyridine

5-Bromopyridin-2-yl-amine (13.8 g, 0.08 mol), acetonylacetone (14.1 ml,0.12 mol) and p-toluenesulphonic acid (100 mg) were dissolved in toluene(180 ml) and refluxed under Dean Stark conditions for 14 hours. Aftercooling, the brown solution was poured into water (200 ml) and extractedwith toluene (2×200 ml). The organic extracts were combined and washedwith brine (50 ml) then dried over anhydrous magnesium sulphate,filtered and concentrated in vacuo to give crude product. This waspurified by column chromatography on silica eluting with ethyl acetate:pentane (1:3) to give the title compound as a brown oil (18.4 g, 0.073mol, 92%). ¹H NMR (CDCl₃, 400 MHz) δ: 2.18 (s, 6H), 5.90 (s, 2H), 7.11(d, 1H), 7.92 (d, 1H), 8.62 (s, 1H). LRMS: m/z 253 (M−H⁺, Br isotope).

EXAMPLE 39 2-Chloro-1-[6-(2,5-dimethylpyrrol-1-yl)pyridin-3-yl]ethanone

To a solution of bromo pyridine from example 38 (2 g, 8.0 mmol) at −78°C., in dry THF (30 ml), was added butyllithium (2.5M in hexanes) (3.5 ml8.8 mmol), dropwise over 20 minutes. The reaction mixture was stirredfor 30 minutes then 2-chloro-N-methoxy-N-methylacetamide (1.2 g, 8.8mmol) in dry THF (20 ml) was added dropwise keeping the temperature at−78° C. Stirring was continued for 30 minutes at this temperature before1 M HCl (aq) (50 ml) was added and the reaction mixture warmed to roomtemperature. The organic layer was separated and the aqueous layerwashed with ethyl acetate (50 ml). The organic layers were combined thenwashed with 3M NaOH (aq) (10 ml) and brine (10 ml) before being driedover anhydrous magnesium sulphate, filtered and concentrated in vacuo togive crude title compound as a brown oil (1.34 g, 5.4 mmol, 67%). ¹H NMR(CDCl₃, 400 MHz) δ: 2.20 (s, 6H), 4.68 (s, 2H), 5.92 (s, 2H), 7.32 (d,1H), 8.38 (d, 1H), 9.16 (s, 1H). LRMS: m/z 249 (M−H⁺).

EXAMPLE 40 2-(2,5-dimethylpyrrol-1-yl)-5-oxiranylpyridine

To the ketone from example 39 (1.34 g, 5.4 mmol) dissolved in dry THF(20 ml), cooled to 0° C., was added sodium borohydride (308 mg, 8.1mmol) portionwise. The reaction mixture was stirred for 2 hours then 3MNaOH (aq) (10 ml) was added and stirring continued for a further 16hours. The reaction mixture was extracted with ethyl acetate (2×20 ml)and the combined organic extracts washed with brine (5 ml), dried overanhydrous magnesium sulphate, filtered and concentrated in vacuo. Theresidue was purified by column chromatography on silica eluting withethyl acetate:pentane (1:5) to give the title compound as a colourlessoil (900 mg, 4.2 mmol, 78%). ¹H NMR (CDCl₃, 400 MHz) δ: 2.13 (s, 6H),2.91 (dd, 1H), 3.25 (t, 1H), 3.98 (t, 1H), 5.90 (s, 2H), 7.20 (d, 1H),7.62 (dd, 1H), 8.58 (s, 1H). LRMS: m/z 215 (M−H⁺).

EXAMPLE 411-[6-(2,5-dimethylpyrrol-1-yl)pyridin-3-yl]-2-propylaminoethanol

To the epoxide from example 40 (900 mg, 4.2 mmol) in DMSO (5 ml) wasadded propylamine (4 ml, 4.8 mmol) and the reaction mixture was heatedto 40° C. for 4 days. The reaction mixture was then cooled and 3M HCl(aq) (10 ml) and water (10 ml) were added before washing with diethylether (2×10 ml). This organic layer was discarded. The aqueous layer wasbasified with NH₄OH (5 ml) and extracted with ethyl acetate (3×10 ml).The organic extracts were combined and dried over anhydrous magnesiumsulphate, filtered and concentrated in vacuo to give the title compoundas an oil (1.15 g, 4.2 mmol, 100%). ¹H NMR (CDCl₃, 400 MHz) δ: 0.93 (t,3H), 1.62 (m, 2H), 2.11 (s, 6H), 2.69-2.82 (m, 3H), 3.06 (dd, 1H), 3.60(bs, 2H), 4.92 (dd, 1H), 5.84 (s, 2H), 7.20 (d, 1H), 7.88 (d, 1H), 8.61(s, 1H). LRMS: m/z 274 (M−H⁺).

EXAMPLE 426-[6-(2,5-dimethylpyrrol-1-yl)pyridin-3-yl]-4-propylmorpholi-3-one

Prepared following the same method as for example 27 with the amine fromexample 41 (1.15 g, 4.2 mmol). Purification by column chromatography onsilica eluting with dichloromethane: methanol (98:2) gave the titlecompound as a brown film (191 mg, 0.61 mmol, 14%). ¹H NMR (CDCl₃, 400MHz) δ: 0.97 (t, 3H), 1.65 (m, 2H), 2.13 (s, 6H), 3.38 (m, 1H),3.42-3.56 (m, 2H), 6.61 (t, 1H), 4.35 (d, 1H), 4.45 (d, 1H), 4.91 (dd,1H), 6.91 (s, 2H), 7.22 (d, 1H), 7.89 (d, 1H), 8.61 (s, 1H). LRMS: m/z314 (M−H⁺).

EXAMPLE 436-[6-(2,5-dimethylpyrrol-1-yl)pyridin-3-yl]-4-propylmorpholine

To a solution of the morpholin-3-one from example 42 (191 mg, 0.61 mmol)in dry THF (5 ml) was added lithium aluminium hydride (1 M solution indiethyl ether) (1.25 ml, 0.61 mmol) and the reaction mixture was warmedto reflux for 2.5 hours. The reaction mixture was cooled to roomtemperature then 1 M NaOH (1.25 ml) was added to give a whiteprecipitate. The reaction mixture was filtered and concentrated invacuo. The white solid was discarded. The concentrated filtrate waspurified by column chromatography on silica eluting withdichloromethane:methanol (95:5) to give the title compound as a whitefilm (108 mg, 0.36 mmol, 59%). ¹H NMR (CDCl₃, 400 MHz) δ: 0.92 (t, 3H),1.61 (q, 2H), 2.10 (s, 6H), 2.15 (m, 1H), 2.29 (dt, 1H), 2.40 (t, 2H),2.82 (d, 1H), 3.02 (d, 1H), 3.90 (t, 1H), 4.08 (d, 1H), 4.71 (d, 1H),5.89 (s, 2H), 7.20 (d, 1H), 7.81 (d, 1H), 8.60 (s, 1H). LRMS: m/z 300(M−H⁺).

EXAMPLES 44A AND 44B 5-(4-propylmorpholin-2-yl)pyridin-2-ylamine

To the 2,5-dimethylpyrrole from example 43 (45 mg, 0.15 mmol) in ethanol(3 ml) was added hydroxylamine hydrochloride (52 mg, 0.75 mmol) and thereaction mixture heated to 80° C. for 20 hours. The reaction mixture wascooled to room temperature and concentrated in vacuo. The residue waspurified by column chromatography on silica eluting withdichloromethane:methanol:ammonia (90:10:1) to give the racemic compoundas a colourless film (31 mg, 0.14 mmol, 94%). ¹H NMR (CDCl₃, 400 MHz) δ:0.92 (t, 3H), 1.60 (m, 2H), 2.11 (t, 1H), 2.25 (dt, 1H), 2.41 (t, 2H),2.82-2.91 (dd, 2H), 3.89 (dt, 1H), 4.01 (dd, 1H), 4.57 (bd, 3H), 6.49(d, 1H), 7.42 (d, 1H), 8.02 (s, 1H). LRMS: m/z 222 (M−H⁺).

A sample of this racemic product (580 mg) was separated into it'sconsituent enantiomers by chiral HPLC

Conditions used: Chiralpak AD column (250×21.2 mm), Eluentmethanol:ethanol (1:1), flow rate 15 mL/min.

The faster eluting enantiomer Example 44A (retention time 8.3 min) wasobtained in>99% ee

¹H NMR (CDCl₃, 400 MHz) was identical to that of the racemate. LRMS: m/z222. Analysis found C, 63.54; H, 8.60; N, 18.38%. C₁₂H₁₉N₃O.3H₂Orequires C, 63.58; H, 8.71; N, 18.53%.

[α]²⁵ ₅₄₆−2.1 (c=0.12, MeOH); [α]²⁵ ₄₃₆ −8.9 (c=0.12, MeOH)

The slower eluting enantiomer, Example 44B (retention time 9.4 min) wasobtained in 98.9% e.e.

¹H NMR (CDCl₃, 400 MHz) was identical to that of the racemate. LRMS: m/z222. Analysis found C, 63.53; H, 8.57; N, 18.36%. C₁₂H₁₉N₃O.3H₂Orequires C, 63.58; H, 8.71; N, 18.53%.

[α]²⁵ ₅₄₆+2.4(c=0.12, MeOH); [α]²⁵ ₄₃₆+7.2 (c=0.12, MeOH)

EXAMPLE 45 2-Ethyl-6-(3-methoxy-phenyl)-4-propyl-morpholin-3-one

Sodium hydroxide (0.48 g, 12.0 mmol) in water (2 mL) was added to theproduct from example 3 (0.50 g, 2.4 mmol) in dichloromethane (5 mL) andthe mixture stirred at room temperature. 2-Chlorobutyryl chloride (0.28mL, 2.87 mmol) was then added dropwise and the reaction mixture stirredfor 60 hours. The reaction mixture was diluted with dichloromethane (10mL) and the aqueous layer was separated. The organic layer was driedover anhydrous magnesium sulphate, filtered and concentrated in vacuo togive the crude product as a clear oil (contained mixture of cyclised anduncyclised material) (0.57 g). LRMS: m/z 314 (M−H⁺ of uncyclisedmaterial), 296 (M−H+ less water), 278 (M−H⁺ of cyclised product).Potassium hydroxide (0.13 g, 2.20 mmol) was dissolved in water (1 mL)and added to a solution of the crude product (0.57 g, 1.83 mmol) inisopropyl alcohol (5 mL). The reaction mixture was stirred at roomtemperature overnight and the organic solvent then evaporated in vacuo.The residue was dissolved in ethyl actetate (10 mL) and the aqueouslayer separated. The organic layer was dried over anhydrous magnesiumsulphate, filtered and concentrated in vacuo to give the crude productas an oil. The residue was purified by column chromatography on silicaeluting with ethyl acetate:pentane (1:5 to 1:1) to give the titlecompound as a clear oil (326 mg, 1.17 mmol, 49%) as a mixture ofdiastereomers. ¹H NMR (CDCl₃, 400 MHz) δ: 0.90 (t, 3H), 1.00 (t, 3H),1.60 (m, 2H), 2.00 (bm, 2H), 3.10-3.60 (m, 4H), 3.80 (s, 3H), 4.20 (d,0.5H), 4.25 (d, 0.5H), 4.75 (d, 0.5H), 4.90 (d, 0.5H), 6.80 (d, 1H),6.90 (m, 2H), 7.25 (m, 1H). LRMS (APCl): m/z 278 (MH⁺), 276 (MH⁻).

EXAMPLES 46A AND 46B 2-Ethyl-6-(3-methoxy-phenyl)-4-propyl-morpholine

Borane-tetrahydrofuran complex (1 M in THF) (3 mL, 3 mmol) was addeddropwise to the product from example 45 (0.33 g, 1.18 mmol) in dry THF(4 mL) under an atmosphere of nitrogen. The reaction mixture was heatedat 85° C. for 3 hours then cooled and quenched by the addition ofmethanol (1 mL). The reaction mixture was then concentrated in vacuo andthe residue suspended in 6N HCl (aq) (10 mL) and heated to 60° C. for1.5 hours. The reaction mixture was cooled and extracted with diethylether (2×10 mL). The aqueous layer was rendered basic (pH 9-10) byaddition of solid potassium carbonate before re-extracting withdichloromethane (2×15 mL). The dichloromethane extracts were dried overanhydrous magnesium sulphate, filtered and concentrated in vacuo to givethe crude products as a clear oil. Purification by column chromatographyon silica eluting with ethyl acetate:pentane (1:10) yielded the twotitle compounds as single diastereomers. Example 46A: clear oil (0.10 g,0.38 mmol, 32%): ¹H NMR (CDCl₃, 400 MHz) δ: 1.00 (m 6H), 1.60 (bm, 3H),1.85 (m, 1H), 2.25 (bt, 2H), 2.35 (s, 1H), 2.45 (m, 1H), 2.60 (m, 1H),2.65 (m, 1H), 3.70 (s, 1H), 3.80 (s, 3H), 4.80 (s, 1H), 6.80 (d, 1H),7.00 (m, 2H), 7.25 (m, 1H). LRMS (APCl): m/z 264 (M−H⁺). Example 46B:clear oil (0.10 g, 0.38 mmol, 32%): ¹H NMR (CDCl₃, 400 MHz) δ: 0.90 (t,3H), 1.00 (t, 3H), 1.60 (bm, 4H), 1.80 (bs, 1H), 2.00 (bs, 1H), 2.35(bs, 2H), 2.85 (bd, 1H), 2.95 (bd, 1H), 3.60 (s, 1H), 3.80 (s, 3H), 4.60(s, 1H), 6.80 (d, 1H), 6.95 (s, 2H), 7.25 (t, 1H). LRMS (APCl): m/z 264(MH⁺).

EXAMPLE 47A 3-(6-Ethyl-4-propyl-morpholin-2-yl)-phenol

Hydrobromic acid (48% aq., 5 mL) and the product from example 46A (0.10g, 0.38 mmol) were heated at 80° C. for 16 hours. After cooling thereaction mixture was concentrated in vacuo. The residue was partitionedbetween aqueous ammonia (0.880, 15 mL) and dichloromethane (15 mL), thelayers were separated and the aqueous layer re-extracted withdichloromethane (2×15 mL). The organic extracts were combined, driedover anhydrous magnesium sulphate, filtered and concentrated in vacuo.The crude product was purified by column chromatography on silicaeluting with dichloromethane, then dichloromethane:methanol (99:1 to95:5) to yield the title compound as a clear oil (65 mg, 0.26 mmol, 69%)as the single diastereoisomer. ¹H NMR (CDCl₃, 400 MHz) δ: 0.95 (m 6H),1.60 (m, 3H), 1.85 (m, 1H), 2.25 (m, 2H), 2.45 (m, 2H), 2.55 (q, 1H),2.75 (d, 1H), 3.75 (s, 1H), 4.80 (m, 1H), 6.70 (d, 1H), 6.90 (s, 1H),7.00 (1H, d), 7.25 (t, 1H). LRMS (APCl): m/z 250 (MH⁺). Analysis foundC, 70.94%; H, 9.16%; N, 5.53%. C₁₅H₂₃NO₂.0.3H₂O requires C, 70.72%; H,9.34%; N, 5.50%.

EXAMPLE 47B 3-(6-Ethyl-4-propyl-morpholin-2-yl)-phenol

Prepared following the same method as for example 47A with the productfrom example 46B (0.10 g, 0.38 mmol). Purification by columnchromatography on silica was not required. The title compound wasobtained as a yellow oil (57 mg, 0.23 mmol, 60%) as the singlediastereoisomer. ¹H NMR (CDCl₃, 400 MHz) δ: 0.90 (t, 3H), 1.00 (t, 3H),1.60 (m, 4H), 1.85 (t, 1H), 2.00 (t, 1H), 2.35 (m, 2H), 2.90 (d, 1H),3.00 (d, 1H), 3.65 (m, 1H), 4.60 (m, 1H), 6.75 (d, 1H), 6.80 (s, 1H),6.90 (1H, d), 7.20 (t, 1H). LRMS (ESI): m/z 250 (MH⁺), 248 (M−H⁻).Analysis found C, 71.63%; H, 9.19%; N, 5.55%. C₁₅H₂₃NO₂.0.1H₂O requiresC, 71.73%; H, 9.31%; N, 5.58%.

EXAMPLE 48 2-Methyl-6-(3-methoxy-phenyl)-4-propyl-morpholin-3-one

Prepared following the same method as for example 45 with the productfrom example 3 (0.44 g, 2.10 mmol) and 2-chloropropionyl chloride (0.25mL, 2.50 mmol). Purification by column chromatography on silica of thetitle compound was not required. The title compound was obtained as aclear oil (0.42 g, 1.60 mmol, 76%) as a mixture of diastereomers. ¹H NMR(CDCl₃, 400 MHz) δ: 0.95 (t, 3H), 1.60 (m, 5H), 3.30 (bm, 2H), 3.50 (bm,2H), 3.80 (s, 3H), 4.40 (q, 0.5H), 4.55 (q, 0.5H), 4.80 (dd, 0.5H), 4.95(dd, 0.5H), 6.85 (d, 1H), 6.95 (s, 2H), 7.25 (m, 1H). LRMS (APCl): m/z264 (MH⁺), 262 (MH⁻).

EXAMPLES 49A AND 49B 2-Methyl-6-(3-methoxy-phenyl)-4-propyl-morpholine

Prepared following the same method as for example 46 with the productfrom example 48 (0.42 g, 1.6 mmol). Purification by columnchromatography on silica eluting with ethyl acetate: pentane (1:6)yielded the two title compounds as single diastereomers.

Example 49A: clear oil (0.10 g, 0.40 mmol, 25%): ¹H NMR (CDCl₃, 400 MHz)δ: 0.95 (t 3H), 1.30 (d, 3H), 1.60 (m, 2H), 2.20-2.35 (m, 3H), 2.50 (d,1H), 2.60 (m, 1H), 2.65 (d, 1H), 3.80 (s, 3H), 4.00 (s, 1H), 4.85 (s,1H), 6.80 (d, 1H), 7.05 (m, 2H), 7.25 (m, 1H). LRMS (APCl): m/z 250(MH⁺).

Example 49B: clear oil (0.10 g, 0.40 mmol, 25%): ¹H NMR (CDCl₃, 400 MHz)δ: 0.90 (t 3H), 1.25 (m, 3H), 1.60 (m, 2H), 1.80 (m, 1H), 2.00 (bm, 1H),2.35 (s, 2H), 2.80 (d, 1H), 2.90 (d, 1H), 3.80 (s, 3H), 3.85 (s, 1H),4.60 (s, 1H), 6.80 (d, 1H), 7.00 (m, 2H), 7.25 (m, 1H). LRMS (APCl): m/z250 (MH⁺).

EXAMPLE 50A 3-(6-Methyl-4-propyl-morpholin-2-yl)-phenol

Prepared following the same method as for example 47A with the productfrom example 49A (0.10 g, 0.4 mmol). Purification by columnchromatography on silica eluting with dichloromethane, thendichloromethane:methanol (99:1) yielded the title compound as a clearoil (70 mg, 0.30 mmol, 74%) as the single diastereoisomer. ¹H NMR(CDCl₃, 400 MHz) δ: 0.95 (t, 3H), 1.35 (d, 3H), 1.55 (m, 2H), 2.25 (m,2H), 2.35 (m, 1H), 2.50 (m, 1H), 2.55 (m, 1H), 2.75 (d, 1H), 4.05 (s,1H), 4.85 (m, 1H), 6.70 (d, 1H), 6.90 (s, 1H), 7.00 (1H, d), 7.20 (t,1H). LRMS (APCl): m/z 236 (MH⁺). Analysis found C, 70.62%; H, 8.89%; N,5.95%. C₁₄H₂₁NO₂.0.1H₂O requires C, 70.91%; H, 9.01%; N, 5.91%.

EXAMPLE 50B 3-(6-Methyl-4-Propyl-morpholin-2-yl)-phenol

Prepared following the same method as for example 47A with the productfrom example 49B (0.10 g, 0.4 mmol). Purification by columnchromatography on silica was not required. The title compound wasobtained as a yellow oil (100 mg, 0.42 mmol, 103%—contained 3% startingmaterial) as the single diastereomer. ¹H NMR (CDCl₃, 400 MHz) δ: 0.90(t, 3H), 1.25 (d, 3H), 1.60 (m, 2H), 1.85 (m, 1H), 2.00 (m, 1H), 2.35(m, 2H), 2.85 (d, 1H), 3.00 (d, 1H), 3.85 (s, 1H), 4.60 (d, 1H), 6.75(d, 1H), 6.80 (s, 1H), 6.90 (1H, d), 7.20 (m, 1H). LRMS (APCl): m/z 236(MH⁺). Analysis found C, 69.38%; H, 8.86%; N, 5.73%. C₁₄H₂₁NO₂.0.45H₂Orequires C, 69.33%; H, 9.06%; N, 5.78%.

EXAMPLE 51 1-(4-Chloro-3-methoxy-phenyl)-2-propylamino-ethanol

Sodium triacetoxyborohydride (1.25 g, 5.89 mmol) was added with care toa solution of 2-amino-1-(4-chloro-3-methoxy-phenyl)-ethanol (J. Med.Chem., 30(10), 1887, (1987)) (600 mg, 2.98 mmol) and propionaldehyde(0.22 mL, 2.96 mmol) in dichloromethane (10 mL), and the reactionmixture was stirred at room temperature for 1 hour. Sodium bicarbonatesolution (sat. aq., 10 mL) was added dropwise and then the reactionmixture was diluted further with water (20 mL) and dichloromethane (20mL). The aqueous layer was separated and re-extracted withdichloromethane (2×20 mL). The combined organic layers were dried overanhydrous magnesium sulphate, filtered and concentrated in vacuo. Thecrude product was purified by column chromatography on silica elutingwith dichloromethane:methanol:0.880 ammonia (95:5:0.5 to 92:8:0.8) toyield the title compound as a solid (320 mg, 1.31 mmol, 44%). ¹H NMR(CDCl₃, 400 MHz) δ: 0.90 (t, 3H), 1.50 (q, 2H), 2.50-2.70 (m, 5H), 2.90(dd, 1H), 3.80 (s, 3H), 4.65 (dd, 1H), 6.85 (d, 1H), 7.00 (1H, d), 7.30(bd, 1H). LRMS (APCl): m/z 244 (MH⁺), 226 (MH⁺ less H₂O).

EXAMPLE 52 6-(4-Chloro-3-methoxy-phenyl)-4-propyl-morpholin-3-one

Chloroacetyl chloride (0.11 mL, 1.33 mmol) was added to a solution ofthe product from example 51 (0.31 g, 1.27 mmol) and triethylamine (0.19mL, 1.36 mmol) in dichloromethane (10 mL) and stirred at roomtemperature for 60 hours. The reaction mixture was diluted withdichloromethane (20 mL) and washed with hydrochloric acid (aq. 1N, 10mL), water (10 mL) and sodium bicarbonate solution (sat. aq., 10 mL).The organic layer was dried over anhydrous magnesium sulphate, filteredand concentrated in vacuo to yield the uncyclised product as an oil(0.40 g). LRMS (APCl): m/z 320 (MH⁺ of uncyclised product), 302 (MH⁺less water), 284 (MH⁺ of cyclised product). Potassium hydroxide (0.75 g,1.33 mmol) was added to a solution of the uncyclised product (0.40 g,1.23 mmol) in isopropyl alcohol (10 mL) and water (0.4 mL) and stirredat room temperature for 16 hours. The reaction mixture was concentratedin vacuo and partitioned between dichloromethane (30 mL) and water (30mL). The layers were separated and the aqueous layer re-extracted withdichloromethane (2×20 mL). The combined organics were washed with water(30 mL), dried over anhydrous magnesium sulphate, filtered andconcentrated in vacuo to yield the title compound as an oil (0.34 g,1.19 mmol, 94%). ¹H NMR (CDCl₃, 400 MHz) δ: 0.95 (t, 3H), 1.60-1.70 (m,2H), 3.30-3.40 (m, 2H), 3.40-3.55 (m, 2H), 3.95 (s, 3H), 4.35 (bd, 1H),4.42 (bd, 1H), 4.78 (dd, 1H), 6.85 (dd, 1H), 7.00 (s, 1H), 7.38 (dd,1H). LRMS (APCl): m/z 284 (MH⁺).

EXAMPLE 53 6-(4-Chloro-3-methoxy-phenyl)-4-propyl-morpholine

Borane-tetrahydrofuran complex (1 M in THF) (3.5 mL, 3.5 mmol) was addeddropwise to a solution of the product from example 52 (0.33 g, 1.16mmol) in dry THF (3 mL) under an atmosphere of nitrogen. The reactionmixture was refluxed for 2.5 hours then cooled and quenched by additionof methanol (1 mL). The reaction mixture was concentrated in vacuo andthe residue suspended in 4N HCl (aq., 8 mL) and refluxed for 2 hours.The reaction mixture was cooled and extracted with dichloromethane (2×10mL). The organic layers were discarded. The aqueous layer was renderedbasic (pH 9-10) by addition of solid potassium carbonate beforere-extracting with dichloromethane (2×15 mL). The dichloromethaneextracts were washed with water (10 mL), dried over anhydrous magnesiumsulphate, filtered and concentrated in vacuo to give the title compoundas an oil (0.31 g, 1.15 mmol, 99%). ¹H NMR (CDCl₃, 400 MHz) δ: 0.95 (t,3H), 1.45-1.60 (m, 2H), 2.00 (t, 1H), 2.20 (t, 1H), 2.35 (t, 2H), 2.80(d, 1H), 2.90 (d, 1H), 3.80 (t, 1H), 3.90 (s, 3H), 4.03 (dd, 1H), 4.55(d, 1H), 6.85 (dd, 1H), 7.00 (s, 1H), 7.30 (dd, 1H). LRMS (APCl): m/z270 (MH⁺).

EXAMPLE 54 2-Chloro-5-(4-propyl-morphlin-2-yl)-phenol

Prepared following the same method as for example 7b (although refluxingwas continued for 2.5 hours rather than 1 hour) with the product fromexample 53 (0.28 g, 1.02 mmol). Purification by column chromatography onsilica was not required. The title compound was yielded as a pale browngum (0.21 g, 0.82 mmol, 81%). ¹H NMR (CDCl₃, 400 MHz) δ: 0.93 (t, 3H),1.55 (q, 2H), 2.0 (t, 1H), 2.20 (dt, 1H), 2.30-2.40 (m, 2H), 2.80 (bd,1H), 2.90 (bd, 1H), 3.80 (dt, 1H), 4.0 (dd, 1H), 4.30 (d, 1H), 6.87 (dt,1H), 7.02 (fd, 1H), 7.25 (s, 1H). LRMS (APCl): m/z 256 (MH⁺). Analysisfound C, 60.71%; H, 7.10%; N, 5.45%. C₁₃H₁₈NO₂Cl requires C, 61.05%; H,7.09%; N, 5.48%.

EXAMPLE 55 Methyl (2S)-2-(Propionylamino)propanoate

L-Alanine methyl ester hydrochloride salt (14 g, 0.1 mol) was dissolvedin dichloromethane (150 mL) and treated with triethylamine (30.45 g, 0.3mmol). The solution was stirred and propionyl chloride added dropwise.After stirring overnight the mixture was quenched by addition of 1 Mhydrochloric acid (200 mL) and the organic layer separated. The aqueouslayer was re-extracted with dichloromethane (3×200 mL) and the combinedorganic layers were dried with magnesium sulfate, filtered andevaporated to a to a clear oil (16.0 g, quant.).

¹H NMR (DMSO-d6, 400 MHz) δ: 0.95 (t, 3H), 1.25 (d, 3H), 2.1 (q, 2H),3.6 (s, 3H), 4.2 (quin, 1H), 8.2 (bd, 1H). LRMS (ESI+) m/z 160 (MH⁺)

EXAMPLE 56 tert-butyl (1S)-2-hydroxy-1-methylethyl(propyl)carbamate

The product from example 55 was dissolved in tetrahydrofuran (200 mL)and borane-tetrahydrofuran complex (300 mL, 0.3 mol) was added to thestirred solution at room temperature. The mixture was then heated atreflux overnight. After to cooling to room temperature, the reaction wasquenched by the cautious addition of 6M hydrochloric acid (100 mL) andthen heated to reflux for 6 hours. The reaction mixture was allowed tocool to room temperature overnight, and then evaporated to dryness(11.77 g). The crude mixture gave m/z 118 consistent with the desiredaminoalcohol intermediate. The crude mixture was then dissolved inmethanol (50 mL) and water (400 mL) before the addition of potassiumhydroxide (28.22 g, 0.5 mol). Di-tert-butyl dicarbonate (32.87 g 0.15mol) was added to the mixture and stirring continued over 3 days. Thereaction mixture was partitioned between DCM (500 mL) and water (100mL), the organic layer separated and the aqueous layer re-extracted withDCM twice more. The combined organic fractions were dried with magnesiumsulfate, filtered and evaporated to a crude. Purification by flashchromatography on SiO₂ eluting with dichloromethane:methanol:880 NH₃(97:3:0.3), afforded the desired product as a clear oil 4.5 g (21%)together with a further 10 g of partially purified material.

¹H NMR (DMSO-d6, 400 MHz) δ: 0.8 (t, 3H), 1.05 (bs, 3H), 1.4 (m, 11H),2.95 (bs, 2H), 3.35 (bm, 3H), 4.6 (bs, 1H) LRMS (ESI+) m/z 240 (MNa⁺)

EXAMPLE 57 (2S)-2-(propylamino)propan-1-ol hydrochloride

The pure material from example 56 (4.2 g, 0.021 mol) was dissolved indioxan (10 mL) and treated with 4M HCl in dioxan (30 mL). The mixturewas stirred at room temperature for 16 hours and then evaporated to awhite solid (2.74 g, 92%)

¹H NMR (DMSO-d6, 400 MHz) δ: 0.9 (t, 3H), 1.15 (d, 3H), 1.6 (m, 2H), 2.8(m, 2H), 3.15 (m, 1H), 3.5 (bm, 1H), 3.6 (m, 1H), 5.4 (bs, 1H), 8.8 (bd,2H). LRMS (APCl+) 118 (MH⁺)

EXAMPLE 58 (5S)-2-(3-methoxyphenyl)-5-methyl-4-propylmorpholin-2-ol

The product from example 57 (1.0 g, 6.6 mmol) was dissolved in toluene(10 mL) and treated with triethylamine (1.38 g, 14 mmol) before theaddition of 2-bromo-3′-methoxyacetophenone (1.5 g, 6.6 mmol). Themixture was heated to 65° C. and stirred over 3 days. After cooling toroom temperature the mixture was partitioned between brine and ethylacetate, the organic layer separated, dried with magnesium sulfate,filtered and evaporated. The residue was purified by flashchromatography on SiO₂ eluting with ethyl acetate, to afford the desiredmorpholinol compound as a mixture of stereoisomers as a pale yellow oil(1.0 g 58%).

¹H NMR (DMSO-d6, 400 MHz) δ: 0.8 (m, 3H), 0.95 (d, 3H), 1.35 (m, 2H),2.1 (m, 2H), 2.4 (bm, 1H), 2.6 (m, 1H), 2.75 (m, 1H), 3.5 (d, 1H), 3.75(m, 4H), 6.0 (s, 0.75H), 6.1 (s, 0.25H), 6.85 (d, 1H), 7.05 (m, 2H),7.25 (t, 1H). LRMS (ESI+) m/z 248 (M−H2O), 266 (MH⁺), 288 (MNa+)

EXAMPLE 59 (5S)-2-(3-methoxyphenyl)-5-methyl-4-propylmorpholine

The product from example 58 (770 mg, 2.9 mmol) was dissolved indichloromethane (20 mL) and cooled to −78° C. under a nitrogenatmosphere. Triethylsilane (3.7 mL, 23 mmol) was added to the stirredmixture followed by trimethylsilyltriflate (1.1 mL, 5.8 mmol). Stirringwas continued overnight and the reaction mixture allowed to reach roomtemperature. The reaction was quenched by the addition of saturatedaqueous sodium bicarbonate solution and extracted with dichloromethane(three times). The combined organic layers were dried with magnesiumsulfate, filtered and evaporated. The crude product was purified byflash chromatography on SiO₂ dichloromethane:methanol:880 ammonia(97:3:0.3), to yield the desired morpholine compound (600 mg, 83%)

¹H NMR (CDCl₃, 400 MHz) δ: 0.95 (m, 3H), 1.1 (b, d, 3H), 1.6 (bm, 2H),2.2-3.1 (5H), 3.5 (bm, 1H), 4.85 (m, 4H), 4.6 (b, 1H), 6.8 (d, 1H), 6.95(m, 2H), 7.25 (m, 1H+CHCl₃)

LRMS (APCl+) m/z 250 (MH⁺)

Analysis found C, 71.53%; H, 9.21%; N, 5.55%. C₁₅H₂₃NO₂.0.15H₂O requiresC, 71.48%; H, 9.32%; N, 5.56%.

EXAMPLE 60 3-[(5S)-5-methyl-4-propylmorpholin-2-yl]phenol

The material from example 59 (400 mg, 1.6 mmol) was dissolved in 48%aqueous hydrobromic acid (8 mL) and the mixture heated to 80° C.overnight. After cooling to room temperature, the mixture was quenchedby the addition of saturated aqueous sodium bicarbonate, and the mixtureextracted with dichloromethane (three times). The combined organiclayers were dried with magnesium sulfate, filtered and evaporated togive the products as a white solid (285 mg, 76%)

¹H NMR (CDCl₃, 400 MHz) δ: 0.9 (m, 3H), 1.1+1.2 (2×d, 3H), 1.5 (m, 2H),2.3 (m, 2H), 2.5 (bm, 1H), 2.8 (bm, 1H), 3.1 (d, 1H), 3.5 (bm, 1H), 3.85(bm, 1H), 4.6 (d, 1H), 6.8 (m, 2H), 6.95 (m, 1H), 7.2 (t, 1H) LRMS(APCl+), 236 (MH⁺)

Analysis found C, 70.61%; H, 9.00%; N, 5.86%. C₁₄H₂₁NO₂.0.1H₂O requiresC, 70.91%; H, 9.01%; N, 5.91%.

This mixture of diasteroisomers was separated on a Chiralcel OJ-H(250*21.2 mm) HPLC column. Mobile phase 100% MeOH, flow rate 15 ml/min.

Sample preparation 200 mg dissolved in 4 ml MeOH, 250 μL injection. Twomajor peaks were obtained, with retention times 5.822 min (example 60A,57 mg 28%) and 7.939 min (example 60B, 12 mg, 6%)

Example 60A: ¹H NMR (CDCl₃, 400 MHz) δ: 0.85 (t, 3H), 1.05 (d, 3H), 1.5(m, 2H+H₂O), 2.2 (m, 2H), 2.4 (m, 1H), 2.8 (m, 1H), 3.0 (d, 1H), 3.4 (t,1H), 3.9 (dd, 1H), 4.55 (d, 1H), 5.6 (bs, 1H), 6.75 (d, 1H), 6.85 (s,1H), 6.95 (d, 1H), 7.2 (t, 1H)

HRMS m/z 236.1643 (MH⁺)

Example 60B: ¹H NMR (CDCl₃, 400 MHz) δ: 0.95 (t, 3H), 1.15 (d, 3H), 1.55(m, 2H), 2.4 (m, 2H), 2.55 (t, 1H), 2.65 (dd, 1H), 2.95 (bm, 1H), 3.8(d, 1H), 3.95 (d, 1H), 4.55 (dd, 1H), 6.75 (d, 1H), 6.85 (s, 1H), 6.95(d, 1H), 7.2 (t, 1H)

HRMS m/z 236.1643 (MH⁺)

EXAMPLE 61 (S)-2-propylamino-propan-1-ol hydrochloride

To (S)-(+)-2-amino-1-propanol (19.6 g, 0.26 mol) dissolved indichloromethane (500 ml) was added propionaldehyde (20.9 ml, 0.28 mol)followed by pre-dried powdered 4A molecular sieves (40 g) and themixture stirred at room temperature overnight. The mixture was filteredthrough a pad of celite, the pad washed with dichloromethane, andsolvent evaporated to give a clear oil. This oil was dissolved inmethanol (200 ml) and NaBH₄ was added portionwise over 15 minutes. Themixture was stirred at room temperature overnight, then quenched bycautious addition of 2M HCl_((aq)) (200 ml), basified by addition of 2MNaOH (200 ml) and methanol removed by evaporation.Di-tert-butyldicarbonate (115 g, 0.52 mol) was added followed by1,4-dioxan (200 ml) and the mixture stirred at room temperatureovernight. 1,4-dioxan was removed by evaporation giving a clear oil. Tothis oil was added 4M HCl in 1,4-dioxan (200 ml) and the mixture stirredat room temperature overnight. The solvent was removed by evaporation togive a white solid (24 g).

¹H NMR (DMSO, 400 MHz) δ: 0.95 (t, 3H), 1.2 (d, 3H), 1.6 (m, 2H), 2.8(m, 2H), 3.15 (m, 1H), 3.5 (bm, 1H), 3.6 (m, 1H), 5.4 (b, 1H), 8.6-8.9(bd, 2H) LRMS (APCl+), 118 (MH⁺)

EXAMPLE 62 (5S)-4-Propyl-5-methylmorpholin-2-one

The material from example 61 (4 g, 26 mmol) was dissolved in benzene,followed by the addition of N-ethyldiisopropylamine (9.07 ml, 52 mmol)and methyl bromoacetate (2.4 ml, 26 mmol). The mixture was heated toreflux with azeotropic removal of water overnight. The solvent wasremoved by evaporation, the crude material dissolved in methanol,pre-absorbed onto SiO₂ and flash chromatographed on SiO₂ eluting with40% EtOAc/Pentane to afford the title morpholinone as a clear oil (1.78g).

¹H NMR (CDCl₃, 400 MHz) δ: 0.9 (t, 3H), 1.1 (d, 3H), 1.5 (m, 2H), 2.25(m, 1H), 2.6 (m, 1H), 2.8 (m, 1H), 3.2 (d, 1H), 3.6 (d, 1H), 4.05 (dd,1H), 4.3 (dd, 1H)

t.l.c. Rf=0.18 (50% EtOAc/Pentane, UV visualisation)

EXAMPLE 63(5S)-2-[6-(2,5-dimethyl-1H-pyrrol-1-yl)pyridin-3-yl]-4-propyl-5-methylmorpholin-2-ol

5-bromo-2-(2,5-dimethyl-pyrrol-1-yl)-pyridine (1.5 g 5.9 mmol) wasazeotroped with toluene and dissolved in THF (20 ml). This mixture wascooled to −78 C. and t-butyllithium (1.7M in pentane, 7 ml, 11.9 mmol)was added maintaining the temperature below −70 C. The material fromexample 62 was dissolved in THF (20 ml) and added to the mixtureimmediately on completion of the t-butyllithium addition. The mixturewas allowed to stir at −78° C. for 30 minutes at which time NH₄Cl (10%aq, 150ml) was added and the mixture extracted into EtOAc (200 ml),dried with magnesium sulphate, filtered and evaporated. Flashchromatography on SiO₂ eluting with a stepped gradient from 25%EtOAc/pentane to 50% EtOAc/pentane gave the title compound as mixture ofdiastereoisomers in approximately 3.5:1 ratio as a yellow oil (480 mg).

¹H NMR (CDCl₃, 400 MHz) (diastereomers) δ: 0.95 (m, 3H), 1.1, 1.2 (2×d,3H) 1.5 (m, 2H), 2.15 (s, 6H), 2.4 (m, 1H), 2.5 (d, 1H), 2.6 (m, 1H),2.75 (m, 1H), 3.85-3.95 (m, 1H), 3.6, 3.75, 4.4 (3×m, 2H), 5.15 (bs,1H), 5.9 (s, 2H), 7.2 (d, 1H), 8.05 (dd, 1H), 8.8 (s, 1H)

LRMS (ES+), 330 (MH⁺), 352 (MNa+)

LRMS (ES−), 328 (M−H)

EXAMPLE 64(2S)-2-[{(2RS)-2-[6-(2,5-dimethyl-1H-pyrrol-1-yl)pyridin-3-yl]-2-hydroxyethyl}propyl)amino]propan-1-ol

(5S)-2-[6-(2,5-dimethyl-1H-pyrrol-1-yl)pyridin-3-yl]-4-propyl-5-methylmorpholin-2-ol(480 mg, 1.45 mmol) was dissolved in ethanol (5 mL) and water (2 mL) andtreated with sodium borohydride (220 mg, 5.8 mmol). The reaction mixturewas left stirring overnight at room temperature before being quenched bythe addition of saturated aqueous NH₄Cl (50 mL) and extracted with ethylacetate (2×100 mL). The organic extracts were combined, dried with MgSO₄and evaporated to give 400 mg of a fluffy white solid which was usedwithout further purification

¹H NMR (CDCl₃, 400 MHz) diastereomers δ: 0.8-1.1 (m, 6H), 1.15, 1.35(2×d, 3H), 1.6-2.0 (m, 2H) 2.1 (s, 6H), 2.5-4.05 (m, 7H), 4.8-5.2 (m,1H), 5.9 (s, 2H), 7.2 (m, 1H), 7.8-8.1 (m, 1H), 8.55 (m, 1H).

LRMS (ES+), 332 (MH⁺)

EXAMPLE 65(2S)-2-[[(2RS)-2-(6-aminopyridin-3-yl)-2-hydroxyethyl](propyl)amino]propan-1-ol

(2S)-2-[[(2RS)-2-(6-aminopyridin-3-yl)-2-hydroxyethyl](propyl)amino]propan-1-ol(400 mg, 1.2 mmol) was dissolved in EtOH (5 mL), hydroxylaminehydrochloride (419 mg, 6 mmol) was added and the mixture heated to 80°C. overnight. The solvent was removed under vacuum and the residuepurified by flash chromatography on SiO₂ eluting withdichloromethane/methanol/880 ammonia (95:5:0.5 increasing polarity to93:7:1) to afford the title compounds as a mixture of diastereoisomers(300 mg, 98%)

¹H NMR (CDCl₃, 400 MHz) (2 diastereomers) δ: 0.82-0.97 (6H, m),2.40-2.77 (2H, m), 3.27-3.51 (2H, m), 4.51 (1H, m), 6.58 (1H, m), 7.49(1H, m), 7.86 (1H, m)

LRMS (APCl+), 254 (MH⁺)

EXAMPLES 66 and 675-[(2S,5S)-5-methyl-4-propylmorpholin-2-yl]pyridin-2-amine and5-[(2R,5S)-5-methyl-4-propylmorpholin-2-yl]pyridin-2-amine

The “diol” from example 65 (300 mg, 1.2 mmol) was dissolved indichloromethane (3 mL), and concentrated sulphuric acid (3 mL) wasadded. The mixture was stirred at room temperature for 3 hours. Thereaction was cooled to 0° C., quenched by the cautious addition of 6Msodium hydroxide solution and then extracted with dichloromethane (4×50mL). The combined extracts were dried (MgSO₄) and evaporated to a browngummy solid. Purification by flash chromatography on SiO₂ eluting with10% methanol in ethyl acetate afforded 5 mg of material enriched in theless polar diastereomer (ca. 80% d.e.), 12 mg of material enriched inthe less polar diastereomer (ca. 80% d.e.) and 150 mg of material ca.1:1 mixture of diastereoisomers (total yield 167 mg, 59%). The latter1:1 mixture was subjected to purification by HPLC using a Chiralpak OD-Hcolumn (250×21.2 mm), eluting with methanol/ethanol (1:1).

The faster eluting diastereoisomer (retention time 8.1 min) was obtainedin >99% d.e (60 mg, 21%). ¹H NMR (CDCl₃, 400 MHz) 0.88 (3H, t), 1.01(3H, d), 1.26 (3H, t), 1.37-1.58 (2H, m), 2.18-2.28 (2H, m), 2.36-2.47(1H, m), 2.69-2.77 (1H, m), 2.90 (1H, m), 3.38 (1H, m), 3.72 (2H, d),3.82 (1H, m), 4.40 (2H, brs), 4.45 (1H, dd), 6.48 (1H, d), 7.45 (1H,dd), 8.04 (1H, d)

LRMS (ES⁺): m/z236 (MH⁺)

[α]²⁵ _(D) 46.28 (c 0.13, MeOH)

The slower eluting diastereoisomer (retention time 10.5 min) wasobtained in >99% d.e. (62 mg, 22%). ¹H NMR (CDCl₃, 400 MHz) 0.93 (3H,t), 1.11 (3H, d), 1.49 (2H, m), 2.38 (2H, m), 2.50-2.56 (2H, m), 2.89(1H, m), 3.75 (1H, m), 3.89 (1H, m), 4.40 (2H, brs), 4.46 (1H, m), 6.50(1H, d), 7.50 (1H, dd), 8.07 (1H, d)

LRMS (ES⁺): m/z236 (MH⁺)

[α]²⁵ _(D) 22.58 (c 0.13, MeOH)

1. 5-[(2R,5S)-5-methyl-4-propylmorpholin-2-yl]pyridin-2-amine or apharmaceutically acceptable salt thereof.